Chemokine-binding protein and methods of use

ABSTRACT

Described herein are chemokine-binding domains of THAP-family polypeptides and pharmaceutical compositions which include a polypeptide comprising a chemokine-binding domain of a THAP-family polypeptide. Also disclosed are methods of binding chemokines, inhibiting the activity of chemokines, detecting chemokines, and reducing the symptoms associated with a chemokine mediated or influenced condition by contacting the chemokine with an agent that includes a polypeptide comprising a chemokine binding domain of a THAP-family polypeptide.

RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. §120 to U.S. patent application Ser. No. 10/601,072, entitledCHEMOKINE-BINDING PROTEIN AND METHODS OF USE, filed Jun. 19, 2003, whichis a continuation-in-part of and claims priority under 35 U.S.C. §120 toU.S. patent application Ser. No. 10/317,832, entitled NOVEL DEATHASSOCIATED PROTEINS, AND THAP1 AND PAR4 PATHWAYS IN APOPTOSIS CONTROL,filed Dec. 10, 2002, which is a nonprovisional application of and claimsbenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/341,997, entitled NOVEL DEATH ASSOCIATED PROTEINS, AND THAP1 ANDPAR4 PATHWAYS IN APOPTOSIS CONTROL, filed Dec. 18, 2001. The disclosureof each of the above-listed priority applications is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to genes and proteins of the THAP(THanatos (death)-Associated Protein) family, and uses thereof. Inparticular, the invention relates to uses of THAP-family proteins, orportions thereof, as chemokine-binding proteins and modulators ofcellular and/or physiological responses.

BACKGROUND

Coordination of cell proliferation and cell death is required for normaldevelopment and tissue homeostasis in multicellular organisms. A defectin the normal coordination of these two processes is a fundamentalrequirement for tumorigenesis.

Progression through the cell cycle is highly regulated, requiring thetransit of numerous checkpoints (for review, see Hunter, 1993). Theextent of cell death is physiologically controlled by activation of aprogrammed suicide pathway that results in morphologically recognizableform of death termed apoptosis (Jacobson et al, 1997; Vaux et al.,1994). Both extra-cellular signals, such as tumor necrosis factor, andintracellular signals, like p53, can induce apoptotic cell death.Although many proteins involved in apoptosis or the cell cycle have beenidentified, the mechanisms by which these two processes are coordinatedare not well understood.

It is well established that molecules which modulate apoptosis have thepotential to treat a wide range of conditions relating to cell death andcell proliferation. For example, such molecules may be used for inducingcell death for the treatment of cancers, inhibiting cell death for thetreatment of neurodegenerative disorders, and inhibiting or inducingcell death for regulating angiogenesis. However, because many biologicalpathways controlling cell cycle and apoptosis have not yet been fullyelucidated, there is a need for the identification of biological targetsfor the development of therapeutic molecules for the treatment of thesedisorders.

PML Nuclear Bodies

PML nuclear bodies (PML-NBs), also known as PODs (PML oncogenicdomains), ND10 (nuclear domain 10) and Kr bodies, are discretesubnuclear domains that are specifically disrupted in cells from acutepromyelocytic leukemia (APL), a distinct subtype of human myeloidleukemia (Maul et al., 2000; Ruggero et al., 2000; Zhong et al., 2000a).Their name derives from their most intensively studied proteincomponent, the promyelocytic leukemia protein (PML), a RING fingerIFN-inducible protein encoded by a gene originally cloned as thet(15;17) chromosomal translocation partner of the retinoic acid receptor(RAR) locus in APL. In APL cells, the presence of the leukemogenicfusion protein, PML-RAR, leads to the disruption of PML-NBs and thedelocalization of PML and other PML-NB proteins into aberrant nuclearstructures (Zhong et al., 2000a). Treatment of both APL cell lines andpatients with retinoic acid, which induces the degradation of thePML-RAR oncoprotein, results in relocalization of PML and other NBscomponents into PML-NBs and complete remission of clinical disease,respectively. The deregulation of the PML-NBs by PML-RAR thus appears toplay a critical role in tumorigenesis. The analysis of mice, where thePML gene was disrupted by homologous recombination, has revealed thatPML functions as a tumor suppressor in vivo (Wang et al., 1998a), thatis essential for multiple apoptotic pathways (Wang et al., 1998b). Pml−/− mice and cells are protected from Fas, TNFα, ceramide andIFN-induced apoptosis as well as from DNA damage-induced apoptosis.However, the molecular mechanisms through which PML modulates theresponse to pro-apoptotic stimuli are not well understood (Wang et al.,1998b; Quignon et al., 1998). Recent studies indicate that PML canparticipate in both p53-dependent and p53-independent apoptosis pathways(Guo et al., 2000; Fogal et al., 2000). p53-dependent DNA-damage inducedapoptosis, transcriptional activation by p53 and induction of p53 targetgenes are all impaired in PML −/− primary cells (Guo et al., 2000). PMLphysically interacts with p53 and acts as a transcriptional co-activatorfor p53. This co-activatory role of PML is absolutely dependent on itsability to recruit p53 in the PML-NBs (Guo et al., 2000; Fogal et al.,2000). The existence of a cross-talk between PML- and p53-dependentgrowth suppression pathways implies an important role for PML-NBs andPML-NBs-associated proteins as modulators of p53 functions. In additionto p53, the pro-apoptotic factor Daxx could be another importantmediator of PML pro-apoptotic activities (Ishov et al., 1999; Zhong etal., 2000b; Li et al., 2000). Daxx was initially identified by itsability to enhance Fas-induced cell death. Daxx interacts with PML andlocalizes preferentially in the nucleus where it accumulates in thePML-NBs (Ishov et al., 1999; Zhong et al., 2000b; Li et al., 2000).Inactivation of PML results in delocalization of Daxx from PML-NBs andcomplete abrogation of Daxx pro-apoptotic activity (Zhong et al.,2000b). Daxx has recently been found to possess strong transcriptionalrepressor activity (Li et al., 2000). By recruiting Daxx to the PML-NBs,PML may inhibit Daxx-mediated transcriptional repression, thus allowingthe expression of certain pro-apoptotic genes.

PML-NBs contain several other proteins in addition to Daxx and p53.These include the autoantigens Sp100 (Stemsdorf et al., 1999) andSp100-related protein Sp140 (Bloch et al., 1999), the retinoblastomatumor suppressor pRB (Alcalay et al., 1998), the transcriptionalco-activator CBP (LaMorte et al., 1998), the Bloom syndrome DNA helicaseBLM (Zhong et al., 1999) and the small ubiquitin-like modifier SUMO-1(also known as sentrin-1 or PIC1; for recent reviews see Yeh et al.,2000; Melchior, 2000; Jentsch and Pyrowblakis, 2000). Covalentmodification of PML by SUMO-1 (sumoylation) appears to play a criticalrole in PML accumulation into NBs (Muller et al., 1998) and therecruitment of other NBs components to PML-NBs (Ishov et al., 1999;Zhong et al., 2000c).

Prostale Apoptosis Response-4

Prostate apoptosis response-4 (PAR4) is a 38 kDa protein initiallyidentified as the product of a gene specifically upregulated in prostatetumor cells undergoing apoptosis (for reviews see Rangnekar, 1998;Mattson et al., 1999). Consistent with an important role of PAR4 inapoptosis, induction of PAR4 in cultured cells is found exclusivelyduring apoptosis and ectopic expression of PAR4 in NIH-3T3 cells(Diaz-Meco et al., 1996), neurons (Guo et al., 1998), prostate cancerand melanoma cells (Sells et al., 1997) has been shown to sensitizethese cells to apoptotic stimuli. In addition, down regulation of PAR4is critical for ras-induced survival and tumor progression (Barradas etal., 1999) and suppression of PAR4 production by antisense technologyprevents apoptosis in several systems (Sells et al., 1997; Guo et al.,1998), including different models of neurodegenerative disorders(Mattson et al., 1999), further emphasizing the critical role of PAR4 inapoptosis. At the carboxy terminus, PAR4 contains both a leucine zipperdomain (Par4LZ, amino acids 290-332), and a partially overlapping deathdomain (Par4DD, amino acids 258-332). Deletion of this carboxy-terminalpart abrogates the pro-apoptotic function of PAR4 (Diaz-Meco et al.,1996; Sells et al., 1997; Guo et al., 1998). On the other hand,overexpression of PAR4 leucine zipper/death domain acts in a dominantnegative manner to prevent apoptosis induced by full-length PAR4 (Sellset al., 1997; Guo et al., 1998). The PAR4 leucine zipper/death domainmediates PAR4 interaction with other proteins by recognizing twodifferent kinds of motifs:zinc fingers of the Wilms tumor suppressorprotein WT1 (Johnstone et al., 1996) and the atypical isoforms ofprotein kinase C (Diaz-Meco et al., 1996), and an arginine-rich domainfrom the death-associated-protein (DAP)-like kinase Dlk (Page et al.,1999). Among these interactions, the binding of PAR4 to aPKCs and theresulting inhibition of their enzymatic activity is of particularfunctional relevance because the aPKCs are known to play a key role incell survival and their overexpression has been shown to abrogate theability of PAR4 to induce apoptosis (Diaz-Meco et al., 1996; Berra etal., 1997).

SLC/CCL21

Chemokine SLC/CCL21 (also known as SLC, CKP-9, 6Ckine, and exodus-2) isa member of the CC (beta)-chemokine subfamily. SLC/CCL21 contains thefour conserved cysteines characteristic of beta chemokines plus twoadditional cysteines in its unusually long carboxyl-terminal domain.Human SLC/CCL21 cDNA encodes a 134 amino acid residue, highly basic,precursor protein with a 23 amino acid residue signal peptide that iscleaved to form the predicted 111 amino acid residues mature protein.Mouse SLC/CCL21 cDNA encodes a 133 amino acid residue protein with 23residue signal peptide that is cleaved to generate the 110 residuemature protein. Human and mouse SLC/CCL21 is highly conserved,exhibiting 86% amino acid sequence identity. The gene for humanSLC/CCL21 has been localized at human chromosome 9p13 rather thanchromosome 17, where the genes of many human CC chemokines areclustered. The SLC/CCL21 gene location is within a region of about 100kb as the gene for MIP-3 beta/ELC/CCL19, another recently identified CCchemokine. SLC/CCL21 was previously known to be highly expressed inlymphoid tissues at the mRNA level, and to be a chemoattractant for Tand B lymphocytes (Nagira, et al. (1997) J. Biol. Chem. 272:19518-19524;Hromas, et al. (1997) J. Immunol. 159:2554-2558; Hedrick, et al. (1997)J. Immunol. 159:1589-1593; Gunn, et al. (1998) Proc. Natl. Acad. Sci.95:258-263). SLC/CCL21 also induces both adhesion of lymphocytes tointercellular adhesion molecule-1 and arrest of rolling cells (Campbell,et al. (1998) Science 279:381-384). All of the above properties areconsistent with a role for SLC/CCL21 in regulating trafficking oflymphocytes through lymphoid tissues. Unlike most CC chemokines,SLC/CCL21 is not chemotactic for monocytes. However, it has beenreported to inhibit hemopoietic progenitor colony formation in adose-dependent manner (Hromas et al. (1997) J. Immunol. 159: 2554-58).

Chemokine SLC/CCL21 is a ligand for chemokine receptor CCR7 (Rossi etal. (1997) J. Immunol. 158:1033; Yoshida et al. (1997) J. Biol. Chem.272:13803; Yoshida et al. (1998) J. Biol. Chem. 273:7118; Campbell etal. (1998) J Cell Biol 141:1053). CCR7 is expressed on T cells anddendritic cells (DC), consistent with the chemotactic action ofSLC/CCL21 for both lymphocytes and mature DC. Both memory (CD45RO⁺) andnaive (CD45RA⁺) CD4⁺ and CD8⁺ T cells express the CCR7 receptor(Sallusto et al. (1999) Nature 401:708). Within the memory T cellpopulation, CCR7 expression discriminates between T cells with effectorfunction that can migrate to inflamed tissues (CCR7-) vs. T cells thatrequire a secondary stimulus prior to displaying effector functions(CCR7⁺) (Sallusto et al. (1999) Nature 401:708). Unlike mature DC,immature DC do not express CCR7 nor do they respond to the chemotacticaction of CCL21 (Sallusto et al. (1998) Eur. J. Immunol. 28:2760; Dieuet al. (1998) J. Exp. Med. 188:373).

A key function of CCR7 and its two ligands SLC/CCL21 and MIP3b/CCL19 isfacilitating recruitment and retention of cells to secondary lymphoidorgans in order to promote efficient antigen exposure to T cells.CCR7-deficient mice demonstrate poorly developed secondary organs andexhibit an irregular distribution of lymphocytes within lymph nodes,Peyer's patches, and splenic periarteriolar lymphoid sheaths (Forster etal. (1999) Cell 99:23). These animals have severely impaired primary Tcell responses largely due to the inability of interdigitating DC tomigrate to the lymph nodes (Forster et al. (1999) Cell 99:23). Theoverall findings to date support the notion that CCR7 and its twoligands, CCL19 and CCL21, are key regulators of T cell responses viatheir control of T cell/DC interactions. CCR7 is an important regulatorymolecule with an instructive role in determining the migration of cellsto secondary lymphoid organs (Forster et al. (1999) Cell 99:23; Nakanoet al. (1998) Blood 91:2886).

SUMMARY OF THE INVENTION

THAP1 (THanatos-Associated-Protein-1)

In the past few years, the inventors have focused on the molecularcharacterization of novel genes expressed in the specialized endothelialcells (HEVECs) of post-capillary high endothelial venules (Girard andSpringer, 1995a; Girard and Springer, 1995b; Girard et al., 1999). Inthe present invention, they report the analysis of THAP1 (for THanatos(death)-Associated Protein-1), a protein that localizes to PML-NBs. Twohybrid screening of an HEVEC cDNA library with the THAP1 bait lead tothe identification of a unique interacting partner, the pro-apoptoticprotein PAR4. PAR4 is also found to accumulate into PML-NBs andtargeting of the THAP1/PAR4 complex to PML-NLs is mediated by PML.Similarly to PAR4, THAP1 is a pro-apoptotic polypeptide. Itspro-apoptotic activity requires a novel protein motif in theamino-terminal part called THAP domain. Together these results define anovel PML-NBs pathway for apoptosis that involves the THAP1/PAR4pro-apoptotic complex.

Embodiments of the present invention include genes, proteins andbiological pathways involved in apoptosis. In some embodiments, thegenes, proteins, and pathways disclosed herein may be used for thedevelopment of polypeptide, nucleic acid or small molecule therapeutics.

One embodiment of the present invention provides a novel protein motif,the THAP domain. The present inventors initially identified the THAPdomain as a 90 residue protein motif in the amino-terminal part of THAP1and which is essential for THAP1 pro-apoptotic activity. THAP1 (THanatos(death) Associated Protein-1), as determined by the present inventors,is a pro-apoptotic polypeptide which forms a complex with thepro-apoptotic protein PAR4 and localizes in discrete subnuclear domainsknown as PML nuclear bodies. However, the THAP domain also defines anovel family of proteins, the THAP family, and the inventors have alsoprovided at least twelve distinct members in the human genome (THAP-0 toTHAP11), all of which contain a THAP domain (typically 80-90 aminoacids) in their amino-terminal part. The present invention thus includesnucleic acid molecules, including in particular the complete cDNAsequences, encoding members of the THAP family, portions thereofencoding the THAP domain or polypeptides homologous thereto, as well asto polypeptides encoded by the THAP family genes. The invention thusalso includes diagnostic and activity assays, and uses in therapeutics,for THAP family proteins or portions thereof, as well as drug screeningassays for identifying compounds capable of inhibiting (or stimulating)pro-apoptotic activity of a THAP family member.

In one example of a THAP family member, THAP1 is determined to be anapoptosis inducing polypeptide expressed in human endothelial cells(HEVECs), providing characterization of the THAP sequences required forapoptosis activity in the THAP1 polypeptide. In further aspects, theinvention is also directed to the interaction of THAP1 with thepro-apoptotic protein PAR4 and with PML-NBs, including methods ofmodulating THAP1/PAR4 interactions for the treatment of disease. Theinvention also concerns interaction between PAR4 and PML-NBs,diagnostics for detection of said interaction (or localization) andmodulation of said interactions for the treatment of disease.

Compounds which modulate interactions between a THAP family member and aTHAP-family target molecule, a THAP domain or THAP-domain targetmolecule, or a PAR4 and a PML-NBs protein may be used in inhibiting (orstimulating) apoptosis of different cell types in various humandiseases. For example, such compounds may be used to inhibit orstimulate apoptosis of endothelial cells in angiogenesis-dependentdiseases including but not limited to cancer, cardiovascular diseases,inflammatory diseases, and to inhibit apoptosis of neurons in acute andchronic neurodegenerative disorders, including but not limited toAlzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateralsclerosis, HIV encephalitis, stroke, epileptic seizures).

Oligonucleotide probes or primers hybridizing specifically with a THAP1genomic DNA or cDNA sequence are also part of the present invention, aswell as DNA amplification and detection methods using said primers andprobes.

Fragments of THAP family members or THAP domains include fragmentsencoded by nucleic acids comprising at least 12, 15, 18, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 consecutivenucleotides selected from the group consisting of SEQ ID NOs: 160-175,or polypeptides comprising at least 8, 10, 12, 15, 20, 25, 30, 40, 50,100, 150 or 200 consecutive amino acids selected from the groupconsisting of SEQ ID NOs: 1 -114.

A further aspect of the invention includes recombinant vectorscomprising any of the nucleic acid sequences described above, and inparticular to recombinant vectors comprising a THAP1 regulatory sequenceor a sequence encoding a THAP1 protein, THAP family member, THAP domain,fragments of THAP family members and THAP domains, homologues of THAPfamily members/THAP domains, as well as to cell hosts and transgenic nonhuman animals comprising said nucleic acid sequences or recombinantvectors.

Another aspect of the invention relates to methods for the screening ofsubstances or molecules that inhibit or increase the expression of theTHAP1 gene or genes encoding THAP family members, as well as withmethods for the screening of substances or molecules that interact withand/or inhibit or increase the activity of a THAP1 polypeptide or THAPfamily polypeptide.

In accordance with another aspect, the present invention provides amedicament comprising an effective amount of a THAP family protein, e.g. THAP1, or a SLC/CCL2 1 -binding fragment thereof, together with apharmaceutically acceptable carrier. The medicaments described hereinmay be useful for treatment and/or prophylaxis.

As related to another aspect, the invention is concerned in particularwith the use of a THAP family protein, homologs thereof and fragmentsthereof, for example THAP1, or a SLC/CCL21-binding fragment thereof asan anti-inflammatory agent. The THAP family protein, for example, THAP1and fragments thereof will be useful for the treatment of conditionsmediated by SLC/CCL21.

In a further aspect, the present invention provides a detection methodcomprising the steps of providing a SLC/CCL21 chemokine-binding moleculewhich is a THAP family protein, for example, THAP1, or anSLC/CCL21-binding fragment thereof, contacting the SLC/CCL21-bindingTHAP1 molecule with a sample, and detecting an interaction of theSLC/CCL21-binding THAP1 molecule with SLC/CCL21 chemokine in the sample.

In one example, the invention may be used to detect the presence ofSLC/CCL21 chemokine in a biological sample. The SLC/CCL21-binding THAP1molecule may be usefully immobilized on a solid support, for example asa THAP1/Fc fusion.

In accordance with another aspect, the present invention provides amethod for inhibiting the activity of SLC/CCL21 chemokine in a sample,which method comprises contacting the sample with an effective amount ofa SLC/CCL21 chemokine-binding molecule which is a THAP1 protein or aSLC/CCL21-binding fragment thereof.

In further aspects the invention provides a purified THAP1 protein or aSLC/CCL21 -binding fragment thereof, or a THAP1/Fc fusion, for use in amethod or a medicament as described herein; and a kit comprising such apurified THAP1 protein or fragment.

Some embodiments of the invention also envisage the use of fragments ofthe THAP1 protein, which fragments have SLC/CCL21 chemokine-bindingproperties. The fragments may be peptides derived from the protein. Useof such peptides can be preferable to the use of an entire protein or asubstantial part of a protein, for example because of the reducedimmunogenicity of a peptide compared to a protein. Such peptides may beprepared by a variety of techniques including recombinant DNA techniquesand synthetic chemical methods.

In addition to the above properties, THAP1 has the capability to bind toseveral additional chemokines. Such chemokines include, but are notlimited to, ELC/CCL19, RANTES CCL5, MIG/CXCL9 and IP10/CXCL10. As such,further aspects of the present invention relate to the binding ofchemokines by THAP1, a chemokine binding domain of THAP1, andpolypeptides having at least 30% amino acid identity to THAP1 or achemokine-binding domain of THAP1. Also contemplated is the binding ofchemokines to oligomers and Fc immunoglobulin fusions of theabove-listed polypeptides.

According to some aspects of the present invention, a THAP1 polypeptide,a chemokine-binding domain of THAP1, polypeptides having at least 30%amino acid identity to THAP1 or a chemokine-binding domain of THAP1 aswell as oligomers or Fc immunoglobulin fusions of these proteins can beused in pharmaceutical compositions and/or medicaments for reducing thesymptoms associated with inflammation and/or inflammatory diseases. Assuch, some aspects of the present invention include pharmaceuticalcompositions and/or medicaments comprising THAP1 protein, achemokine-binding domain of THAP1, polypeptides having at least 30%amino acid identity to THAP1 or a chemokine-binding domain of THAP1 aswell as oligomers or Fc immunoglobulin fusions of these proteins.

Yet other aspects of the invention relate THAP-family polypeptides,chemokine binding domains of THAP-family peptides, fusions of aTHAP-family polypeptide with an immunoglobulin Fc region, fusions of achemokine-binding domain of a THAP-family peptide with an immunoglobulinFc region, oligomers of THAP family polypeptides, chemokine-bindingdomains of THAP family peptides, THAP-family peptide-Fc fusions, andchemokine-binding domain of THAP-family peptide-Fc fusions as well aspolypeptides having at least 30% amino acid identity to any of theabove-listed polypeptides. Pharmaceutical compositions which include oneor more of these polypeptides are also contemplated.

Aspects of the invention relate to methods of binding a chemokine,inhibiting the activity of a chemokine, reducing or ameliorating thesymptoms of a condition mediated or influenced by one or morechemokines, preventing the symptoms of a condition mediated orinfluenced by one or more chemokines and detecting a chemokine by usinga chemokine-binding agents such as THAP-family polypeptides, chemokinebinding domains of THAP-family peptides, fusions of a THAP-familypolypeptide with an immunoglobulin Fc region, fusions of achemokine-binding domain of a THAP-family peptide with an immunoglobulinFc region, oligomers of THAP family polypeptides, chemokine-bindingdomains of THAP family peptides, THAP-family peptide-Fc fusions, andchemokine-binding domain of THAP-family peptide-Fc fusions as well aspolypeptides having at least 30% amino acid identity to any of theabove-listed polypeptides.

It will also be evident that the THAP-family proteins for use-in theinvention may be prepared in a variety of ways, in particular asrecombinant proteins in a variety of expression systems. Any standardsystems may be used such as baculovirus expression systems or mammaliancell line expression systems.

Other aspects of the invention are described in the following numberedparagraphs:

1. A method of identifying a candidate modulator of apoptosiscomprising:

(a) contacting a THAP-family polypeptide or a biologically activefragment thereof with a test compound, wherein said THAP-familypolypeptide comprises at least 30% amino acid identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-114; and

(b) determining whether said compound selectively modulates the activityof said polypeptide; wherein a determination that said test compoundselectively modulates the activity of said polypeptide indicates thatsaid compound is a candidate modulator of apoptosis.

2. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 3, or a biologicallyactive fragment thereof.

3. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 4, or a biologicallyactive fragment thereof.

4. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 5, or a biologicallyactive fragment thereof.

5. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 6, or a biologicallyactive fragment thereof.

6. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 7, or a biologicallyactive fragment thereof.

7. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 8, or a biologicallyactive fragment thereof.

8. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 9, or a biologicallyactive fragment thereof.

9. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 10, or a biologicallyactive fragment thereof.

10. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 11, or a biologicallyactive fragment thereof.

11. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 12, or a biologicallyactive fragment thereof.

12. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 13, or a biologicallyactive fragment thereof.

13. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 14, or a biologicallyactive fragment thereof.

14. The method of Paragraph 1, wherein the THAP-family polypeptidecomprises the amino acid sequence selected from the group consisting ofSEQ ID NOs: 15-114, and biologically active fragments thereof.

15. The method of Paragraph 1, wherein said biologically active fragmentof said THAP-family protein has at least one biological activityselected from the group consisting of interaction with a THAP-familytarget protein, binding to a nucleic acid sequence, binding to PAR-4,binding to PML, binding to a polypeptide found in PML-NBs, localizationto PML-NBs, targeting a THAP-family target protein to PML-NBs, andinducing apoptosis.

16. The methods of any one of Paragraphs 2-15 wherein said THAP-familypolypeptide has at least one biological activity selected from the groupconsisting of interaction with a THAP-family target protein, binding toa-nucleic acid sequence, binding to PAR-4, binding to PML, binding to apolypeptide found in PML-NBs, localization to PML-NBs, targeting aTHAP-family target protein to PML-NBs, and inducing apoptosis.

17. An isolated nucleic acid encoding a polypeptide having apoptoticactivity, said polypeptide consisting essentially of an amino acidsequence selected from the group consisting of:

(a) amino acid positions 1-90 of SEQ ID NO: 2, a fragment thereof havingapoptotic activity, or a polypeptide having at least 30% amino acididentity thereto;

(b) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 89 of SEQ ID NO: 3, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto;

(c) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 89 of SEQ ID NO: 4, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto

(d) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 89 of SEQ ID NO: 5, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto;

(e) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 6, a fragment thereofhaving apoptotic activity or a polypeptide having at least 30% aminoacid identity thereto;

(f) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 7, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto

(g) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 8, a fragment thereofhaving apoptotic activity; or a polypeptide having at least 30% aminoacid identity thereto;

(h) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 9, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto;

(i) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 92 of SEQ ID NO: 10, a fragment thereofhaving apoptotic activity or a polypeptide having at least 30% aminoacid identity thereto;

(j) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 11, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto;

(k) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 12, or a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto;

(l) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 13, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto and

(m) a polypeptide comprising a THAP-family domain consisting essentiallyof amino acid positions 1 to 90 of SEQ ID NO: 14, a fragment thereofhaving apoptotic activity, or a polypeptide having at least 30% aminoacid identity thereto.

18. An isolated nucleic acid encoding a THAP-family polypeptide havingapoptotic activity selected from the group consisting of:

(i) a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of a sequence selected from the group consisting of SEQ IDNOs: 1-114;

(ii) a nucleic acid molecule comprising the nucleic acid sequence of asequence selected from the group consisting of SEQ ID NOs: 160-175 andthe sequences complementary thereto; and

(iii) a nucleic acid the sequence of which is degenerate as a result ofthe genetic code to the sequence of a nucleic acid as defined in (i) and(ii).

19. The nucleic acid of Paragraph 18, wherein said nucleic acidcomprises a nucleic acid selected from the group consisting of SEQ IDNOs. 5, 7, 8 and 11.

20. The nucleic acid of Paragraph 18, wherein said nucleic acidcomprises a nucleic acid selected from the group consisting of SEQ IDNOs. 162, 164, 165 and 168.

21. An isolated nucleic acid encoding a THAP-family polypeptide havingapoptotic activity comprising:

(i) the nucleic acid sequence of SEQ ID NOs: 1-2 or the sequencecomplementary thereto; or

(ii) a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of SEQ ID NOs 1-2;

22. An isolated nucleic acid, said nucleic acid comprising a nucleotidesequence encoding:

i) a polypeptide comprising an amino acid sequence having at least about80% identity to a sequence selected from the group consisting of thepolypeptides of SEQ ID NOs: 1-114 and the polypeptides encoded by thenucleic acids of SEQ ID NOs: 160-175 or

ii) a fragment of said polypeptide which possesses apoptotic activity.

23. The nucleic acid of Paragraph of Paragraph 23, wherein said nucleicacid encodes a polypeptide comprising an amino acid sequence having atleast about 80% identity to a sequence selected from the groupconsisting of the polypeptides of SEQ ID NOs: 5, 7, 8 and 11 and thepolypeptides encoded by the nucleic acids of SEQ ID NOs: 162, 164, 165and 168 or a fragment of said polypeptide which possesses apoptoticactivity.

24. The nucleic acid of Paragraph 23, wherein said polypeptide comprisesan amino acid sequence selected from the group consisting of thesequences of SEQ ID NOs: 5, 7, 8 and 11 and the polypeptides encoded bythe nucleic acids of SEQ ID NOs: 162, 164, 165 and 168.

25. The nucleic acid of Paragraph 23, wherein polypeptide identity isdetermined using an algorithm selected from the group consisting ofXBLAST with the parameters score=50 and wordlength=3, Gapped BLAST withthe default parameters of XBLAST, and BLAST with the default parametersof XBLAST.

26. The nucleic acid of Paragraph 17, wherein said nucleic acid isoperably linked to a promoter.

27. An expression cassette comprising the nucleic acid of Paragraph 26.

28. A host cell comprising the expression cassette of Paragraph 27.

29. A method of making a THAP-family polypeptide, said method comprisingproviding a population of host cells comprising a recombinant nucleicacid encoding said THAP-family protein of any one of SEQ ID NOs. 1-114;and

culturing said population of host cells under conditions conducive tothe expression of said recombinant nucleic acid;

whereby said polypeptide is produced within said population of hostcells.

30. The method of Paragraph 29 wherein said providing step comprisesproviding a population of host cells comprising a recombinant nucleicacid encoding said THAP-family protein of any one of SEQ ID NOs. 5, 7, 8and 11.

31. The method of Paragraph 29, further comprising purifying saidpolypeptide from said population of cells.

32. An isolated THAP polypeptide encoded by the nucleic acid of any oneof SEQ ID Nos. 160-175.

33. The polypeptide of Paragraph 32, wherein said polypeptide is encodedby a nucleic acid selected from the group consisting of SEQ ID NOs. 5,7, 8, 11, 162, 164, 165 and 168.

34. The polypeptide of Paragraph 32, wherein said polypeptide has atleast one activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis.

35. An isolated THAP polypeptide or fragment thereof, said polypeptidecomprising at least 12 contiguous amino acids of a sequence selectedfrom the group consisting of SEQ ID NOs: 1-114.

36. The polypeptide of Paragraph 35, wherein said polypeptide comprisesat least 12 contiguous amino acids of a sequence selected from the groupconsisting of SEQ ID NOs. 5,7, 8,and 11.

37. The polypeptide of Paragraph 35, wherein said polypeptide has atleast one activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis.

38. An isolated THAP polypeptide or fragment thereof, said polypeptidecomprising an amino acid sequence having at least about 80% amino acidsequence identity to a sequence selected from the group consisting ofSEQ ID NOs: 1-114 or a fragment thereof, said polypeptide or fragmentthereof having at least one activity selected from the group consistingof interaction with a THAP-family target protein, binding to a nucleicacid sequence, binding to PAR-4, binding to PML, binding to apolypeptide found in PML-NBs, localization to PML-NBs, targeting aTHAP-family target protein to PML-NBs, and inducing apoptosis.

39. The polypeptide of Paragraph 38, wherein said THAP polypeptide orfragment thereof comprises an amino acid sequence having at least about80% amino acid sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 5, 7, 8 and 11 or a fragment thereof having atleast one activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis.

40. The polypeptide of Paragraph 38, wherein said polypeptide isselectively bound by an antibody raised against an antigenicpolypeptide, or antigenic fragment thereof, said antigenic polypeptidecomprising the polypeptide of any one of SEQ ID NOs: 1-114.

41. The polypeptide of Paragraph 38, wherein said polypeptide isselectively bound by an antibody raised against an antigenicpolypeptide, or antigenic fragment thereof, said antigenic polypeptidecomprising the polypeptide of any one of SEQ ID NOs: 5,7,8 and 11.

42. The polypeptide of Paragraph 38, wherein said polypeptide comprisesthe polypeptide of SEQ ID NOs: 1-114.

43. The polypeptide of Paragraph 38, wherein said polypeptide comprisesa polypeptide selected from the group consisting of SEQ ID NOs. 5, 7, 8and 11.

44. An antibody that selectively binds to the polypeptide of Paragraph38.

45. An antibody according to Paragraph 44, wherein said antibody iscapable of inhibiting binding of said polypeptide to a THAP-familytarget polypeptide.

46. An antibody according to Paragraph 44, wherein said antibody iscapable of inhibiting apoptosis mediated by said polypeptide.

47. The polyptide of Paragraph 38, wherein identity is determined usingan algorithm selected from the group consisting of XBLAST with theparameters score=50 and wordlength=3, Gapped BLAST with the defaultparameters of XBLAST, and BLAST with the default parameters of XBLAST.

48. A method of assessing the biological activity of a THAP-familypolypeptide comprising:

(a) providing a THAP-family polypeptide or a fragment thereof; and

(b) assessing the ability of the THAP-family polypeptide to induceapoptosis of a cell.

49. A method of assessing the biological activity of a THAP-familypolypeptide comprising:

(a) providing a THAP-family polypeptide or a fragment thereof; and

(b) assessing the DNA binding activity of the THAP-family polypeptide.

50. The method of Paragraphs 48 or 49, wherein step (a) comprisesintroducing to a cell a recombinant vector comprising a nucleic acidencoding a THAP-family polypeptide.

51. The method of Paragraphs 49 or 50, wherein the THAP-familypolypeptide comprises a THAP consensus amino acid sequence depicted inSEQ ID NOs: 1-2, or a fragment thereof having at least one activityselected from the group consisting of interaction with a THAP-familytarget protein, binding to a nucleic acid sequence, binding to PAR-4,binding to PML, binding to a polypeptide found in PML-NBs, localizationto PML-NBs, targeting a THAP-family target protein to PML-NBs, andinducing apoptosis.

52. The method of Paragraph 49, wherein the THAP-family polypeptidecomprises an amino acid sequence selected from the group of sequencesconsisting of SEQ ID NOs: 1-114 or a fragment thereof having at leastone activity selected from the group consisting of interaction with aTHAP-family target protein, binding to a nucleic acid sequence, bindingto PAR-4, binding to PML, binding to a polypeptide found in PML-NBs,localization to PML-NBs, targeting a THAP-family target protein toPML-NBs, and inducing apoptosis.

53. The method of Paragraph 49, wherein the THAP-family polypeptidecomprises a native THAP-family polypeptide, or a fragment thereof havingat least one activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis.

54. The method of Paragraph 49, wherein the THAP-family polypeptidecomprises a THAP-family polypeptide or a fragment thereof having atleast one activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis, wherein said THAP-family polypeptideor fragment thereof comprises at least one amino acid deletion,substitution or insertion.

55. An isolated THAP-family polypeptide comprising an amino acidsequence of SEQ ID NOs: 1-114, wherein said polypeptide comprises atleast one amino acid deletion, substitution or insertion with respect tosaid amino acid sequence of SEQ ID NOs. 1-114.

56. A THAP-family polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-114, wherein said polypeptidecomprises at least one amino acid deletion, substitution or insertionwith respect to said amino acid sequence of one of SEQ ID NOs. 1-114 anddisplays a reduced ability to induce apoptosis or bind DNA compared tothe wild-type polypeptide.

57. A THAP-family polypeptide comprising an amino acid sequence of SEQID NOs: 1-114, wherein said polypeptide comprises at least one aminoacid deletion, substitution or insertion with respect to said amino acidsequence of one of SEQ ID NOs. 1-114 and displays a increased ability toinduce apoptosis or bind DNA compared to the wild-type polypeptide.

58. A method of determining whether a THAP-family polypeptide isexpressed within a biological sample, said method comprising the stepsof:

(a) contacting a biological sample from a subject with:

a polynucleotide that hybridizes under stringent conditions to a nucleicacid of SEQ ID NOs: 160-175 or

a detectable polypeptide that selectively binds to the polypeptide ofSEQ ID NOs: 1-114; and

(b) detecting the presence or absence of hybridization between saidpolynucleotide and an RNA species within said sample, or the presence orabsence of binding of said detectable polypeptide to a polypeptidewithin said sample;

wherein a detection of said hybridization or of said binding indicatesthat said THAP-family polypeptide is expressed-within said sample.

59. The method of Paragraph 58, wherein said subject suffers from, issuspected of suffering from, or is susceptible to a cell proliferativedisorder.

60. The method of Paragraph 59, wherein said cell proliferative disorderis a disorder related to regulation of apoptosis.

61. The method of Paragraph 58, wherein said polynucleotide is a primer,and wherein said hybridization is detected by detecting the presence ofan amplification product comprising said primer sequence.

62. The method of Paragraph 58, wherein said detectable polypeptide isan antibody.

63. A method of assessing THAP-family activity in a biological sample,said method comprising the steps of:

(a) contacting a nucleic acid molecule comprising a binding site for aTHAP-family polypeptide with:

-   -   (i) a biological sample from a subject or    -   (ii) a THAP-family polypeptide isolated from a biological sample        from a subject, the polypeptide comprising the amino acid        sequences of one of SEQ ID NOs: 1-114; and

(b) assessing the binding between said nucleic acid molecule and aTHAP-family polypeptide

wherein a detection of decreased binding compared to a referenceTHAP-family nucleic acid binding level indicates that said samplecomprises a deficiency in THAP-family activity.

64. A method of determining whether a mammal has an elevated or reducedlevel of THAP-family expression, said method comprising the steps of:

(a) providing a biological sample from said mammal; and

(b) comparing the amount of a THAP-family polypeptide of SEQ ID NOs:1-114 or of a THAP-family RNA species encoding a polypeptide of SEQ IDNOs: 1-114 within said biological sample with a level detected in orexpected from a control sample;

wherein an increased amount of said THAP-family polypeptide or saidTHAP-family RNA species within said biological sample compared to saidlevel detected in or expected from said control sample indicates thatsaid mammal has an elevated level of THAP-family expression, and whereina decreased amount of said THAP-family polypeptide or said THAP-familyRNA species within said biological sample compared to said leveldetected in or expected from said control sample indicates that saidmammal has a reduced level oF THAP-family expression.

65. The method of Paragraph 64, wherein said mammal suffers from, issuspected of suffering from, or is susceptible to a cell proliferativedisorder.

66. A method of identifying a candidate inhibitor of a THAP-familypolypeptide, a candidate inhibitor of apoptosis, or a candidate compoundfor the treatment of a cell proliferative disorder, said methodcomprising:

(a) contacting a THAP-family polypeptide according to SEQ ID NOs: 1-114or a fragment comprising a contiguous span of at least 6 contiguousamino acids of a polypeptide according to SEQ ID NOs: 1-114 with a testcompound; and

(b) determining whether said compound selectively binds to saidpolypeptide;

wherein a determination that said compound selectively binds to saidpolypeptide indicates that said compound is a candidate inhibitor of aTHAP-family polypeptide, a candidate inhibitor of apoptosis, or acandidate compound for the treatment of a cell proliferative disorder.

67. A method of identifying a candidate inhibitor of apoptosis, acandidate compound for the treatment of a cell proliferative disorder,or a candidate inhibitor of a THAP-family polypeptide of SEQ ID NOs:1-114 or a fragment comprising a contiguous span of at least 6contiguous amino acids of a polypeptide according to SEQ ID NOs: 1-114,said method comprising:

(a) contacting said THAP-family polypeptide with a test compound; and

(b) determining whether said compound selectively inhibits at least onebiological activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis;

wherein a determination that said compound selectively inhibits said atleast one biological activity of said polypeptide indicates that saidcompound is a candidate inhibitor of a THAP-family polypeptide, acandidate inhibitor of apoptosis, or a candidate compound for thetreatment of a cell proliferative disorder.

68. A method of identifying a candidate inhibitor of apoptosis, acandidate compound for the treatment of a cell proliferative disorder,or a candidate inhibitor of a THAP-family polypeptide of SEQ ID NOs:1-114 or a fragment comprising a contiguous span of at least 6contiguous amino acids of a polypeptide according to SEQ ID NOs: 1-114,said method comprising:

(a) contacting a cell comprising said THAP-family polypeptide with atest compound; and

(b) determining whether said compound selectively inhibits at least onebiological activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis;

wherein a determination that said compound selectively inhibits said atleast one biological activity of said polypeptide indicates that saidcompound is a candidate inhibitor of a THAP-family polypeptide, acandidate inhibitor of apoptosis, or a candidate compound for thetreatment of a cell proliferative disorder.

69. The method of Paragraphs 67 or 68, wherein step (b) comprisesassessing apoptotic activity, and wherein a determination that saidcompound inhibits apoptosis indicates that said compound is a candidateinhibitor of said THAP-family polypeptide.

70. The method of Paragraph 68 comprising introducing a nucleic acidcomprising the nucleotide sequence encoding said THAP-family polypeptideaccording to any one of Paragraphs 32-43 into said cell.

71. A polynucleotide according to any one of Paragraphs 17-25 attachedto a solid support.

72. An array of polynucleotides comprising at least one polynucleotideaccording to Paragraph 71.

73. An array according to Paragraph 72, wherein said array isaddressable.

74. A polynucleotide according to any one of Paragraphs 17 to 25 furthercomprising a label.

75. A method of identifying a candidate activator of a THAP-familypolypeptide, said method comprising:

a) contacting a THAP-family polypeptide according to SEQ ID NOs: 1-114or a fragment comprising a contiguous span of at least 6 contiguousamino acids of a polypeptide according to SEQ ID NOs: 1-114 with a testcompound; and

b) determining whether said compound selectively binds to saidpolypeptide;

wherein a determination that said compound selectively binds to saidpolypeptide indicates that said compound is a candidate activator ofsaid polypeptide.

76. A method of identifying a candidate activator of a THAP-familypolypeptide of SEQ ID NOs: 1-114 or a fragment comprising a a contiguousspan of at least 6 contiguous amino acids of a polypeptide according toSEQ ID NOs: 1-114, said method comprising:

(a) contacting said polypeptide with a test compound; and

(b) determining whether said compound selectively activates at least onebiological activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis;

wherein a determination that said compound selectively activates said atleast one biological activity of said polypeptide indicates that saidcompound is a candidate activator of said polypeptide.

77. A method of identifying a candidate activator of a THAP-familypolypeptide of SEQ ID NOs: 1-114 or, a fragment comprising a acontiguous span of at least 6 contiguous amino acids of a polypeptideaccording to SEQ ID NOs: 1-114, said method comprising:

(a) contacting a cell comprising said THAP-family polypeptide with atest compound; and

(b) determining whether said compound selectively activates at least onebiological activity selected from the group consisting of interactionwith a THAP-family target protein, binding to a nucleic acid sequence,binding to PAR-4, binding to PML, binding to a polypeptide found inPML-NBs, localization to PML-NBs, targeting a THAP-family target proteinto PML-NBs, and inducing apoptosis;

wherein a determination that said compound selectively activates said atleast one biological activity of said polypeptide indicates that saidcompound is a candidate activator of said polypeptide.

78. The method of Paragraphs 76 or 77, wherein said determining stepcomprises assessing apoptotic activity, and wherein a determination thatsaid compound increases apoptosis activity indicates that said compoundis a candidate activator of said THAP-family polypeptide.

79. The method of Paragraph 77 wherein step a) comprises introducing anucleic acid comprising the nucleotide sequence encoding saidTHAP-family polypeptide according to any one of Paragraphs 17-25 intosaid cell. 80. A method of identifying a candidate modulator of PAR4activity, said method comprising:

(a) providing a PAR4 polypeptide or a fragment thereof; and

(b) providing a PML-NB polypeptide, or a polypeptide associated withPML-NBs, or a fragment thereof; and

(c) determining whether a test compound selectively modulates theability of said PAR4 polypeptide to bind to said PML-NB polypeptide orpolypeptide associated with PML-NBs;

wherein a determination that said test compound selectively inhibits theability of said PAR4 polypeptide to bind to said PML-NB polypeptide orpolypeptide associated with PML-NBs indicates that said compound is acandidate modulator of PAR4 activity.

81. A method of identifying a candidate modulator of PAR4 activity, saidmethod comprising:

(a) providing a PAR4 polypeptide or a fragment thereof; and

(b) determining whether a test compound selectively modulates theability of said PAR4 polypeptide to localise in PML-NBs;

wherein a determination that said test compound selectively inhibits theability of said PAR4 polypeptide to localise in PML-NBs indicates thatsaid compound is a candidate modulator of PAR4 activity.

82. A method of identifying a candidate inhibitor of THAP-familyactivity, said method comprising:

(a) providing a THAP-family polypeptide of SEQ ID NOs: 1-114 or, afragment comprising a a contiguous span of at least 6 contiguous aminoacids of a polypeptide according to SEQ ID NOs: 1-114; and

(b) providing a THAP-family target polypeptide or a fragment thereof;and

(c) determining whether a test compound selectively inhibits the abilityof said THAP-family polypeptide to bind to said THAP-family targetpolypeptide;

wherein a determination that said test compound selectively inhibits theability of said THAP-family polypeptide to bind to said THAP-familytarget polypeptide indicates that said compound is a candidate inhibitorof THAP-family activity.

83. The method of Paragraph 82, comprising providing a cell comprising:

(a) a first expression vector comprising a nucleic acid encoding aTHAP-family polypeptide of SEQ ID NOs: 1-114 or, a fragment comprising aa contiguous span of at least 6 contiguous amino acids of a polypeptideaccording to SEQ ID NOs: 1-114; and

(b) a second expression vector comprising a nucleic acid encoding aTHAP-family target polypeptide, or a fragment thereof.

84. The method of Paragraph 82, wherein said THAP-family activity isapoptosis activity.

85. The method of Paragraph 82, wherein said THAP-family target proteinis PAR-4.

86. The method of Paragraph 82, wherein said THAP-family polypeptide isa THAP-1, THAP-2 or THAP-3 protein and said THAP-family target proteinis PAR-4.

87. A method of modulating apoptosis in a cell comprising modulating theactivity of a THAP-family protein.

88. The method of Paragraph 87, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

89. A method of modulating apoptosis in a cell comprising modulating therecruitment of PAR-4 to a PML nuclear body.

90. The method of Paragraph 89 wherein modulating the activity of aTHAP-family protein comprises modulating the interaction of aTHAP-family protein and a THAP-family target protein.

91. The method of Paragraph 89 wherein modulating the activity of aTHAP-family protein comprises modulating the interaction of aTHAP-family protein and a PAR4 protein.

92. The method of Paragraph 91 comprising modulation the interactionbetween a THAP-1, THAP-2, or THAP-3 protein and a PAR-4 protein.

93. A method of modulating the recruitment of PAR-4 to a PML nuclearbody comprising modulating the interaction of said PAR-4 protein and aTHAP-family protein.

94. The method of Paragraph 93, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

95. A method of modulating angiogenesis in an individual comprisingmodulating the activity of a THAP-family protein in said individual.

96. The method of Paragraph 95, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

97. A method of preventing cell death in an individual comprisinginhibiting the activity of a THAP-family protein in said individual.

98. The method of Paragraph 97, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

99. The method according to Paragraph 97, wherein the activity of saidTHAP-family protein is inhibited in the CNS.

100. A method of inducing angiogenesis in an individual comprisinginhibiting the activity of a THAP-family protein in said individual.

101. The method of Paragraph 100, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

102. A method according to Paragraph 100, wherein the activity of saidTHAP-family protein is inhibited in endothelial cells.

103. A method of inhibiting angiogenesis or treating cancer in anindividual comprising increasing the activity of a THAP-family proteinin said individual.

104. The method of Paragraph 103, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

105. A method of treating inflammation or an inflammatory disorder in anindividual comprising increasing the activity of a THAP-family proteinin said individual.

106. The method of Paragraph 105, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

107. A method according to Paragraphs 103 or 105, wherein the activityof said THAP-family protein is increased in endothelial cells.

108. A method of treating cancer in an individual comprising increasingthe activity of a THAP-family protein in said individual.

109. The method of Paragraph 108, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs. 1-114.

110. The method of Paragraph 108, wherein increasing the activity ofsaid THAP family protein induces apoptosis, inhibits cell division,inhibits metastatic potential, reduces tumor burden, increasessensitivity to chemotherapy or radiotherapy, kills a cancer cell,inhibits the growth of a cancer cell, kills an endothelial cell,inhibits the growth of an endothelial cell, inhibits angiogenesis, orinduces tumor regression.

111. A method according to any one of Paragraphs 87-110, comprisingcontacting said subject with a recombinant vector encoding a THAP-familyprotein according to any one of Paragraphs 32-43 operably linked to apromoter that functions in said cell.

112. The method of Paragraph 111, wherein said promoter functions in anendothelial cell.

113. A viral composition comprising a recombinant viral vector encodinga THAP-family protein according to Paragraphs 32-43.

114. The composition of Paragraph 113, wherein said recombinant viralvector is an adenoviral, adeno-associated viral, retroviral, herpesviral, papilloma viral, or hepatitus B viral vector.

115. A method of obtaining a nucleic acid sequence which is recognizedby a THAP-family polypeptide comprising contacting a pool of randomnucleic acids with said THAP-family polypeptide or a portion thereof andisolating a complex comprising said THAP-family polypeptide and at leastone nucleic acid from said pool.

116. The method of Paragraph 115 wherein said pool of nucleic acids arelabeled.

117. The method of Paragraph 116 wherein said complex is isolated byperforming a gel shift analysis.

118. A method of identifying a nucleic acid sequence which is recognizedby a THAP-family polypeptide comprising:

(a) incubating a THAP-family polypeptide with a pool of labeled randomnucleic acids;

(b) isolating a complex between said THAP-family polypeptide and atleast one nucleic acid from said pool;

(c) performing an amplification reaction to amplify the at least onenucleic acid present in said complex;

(d) incubating said at least one amplified nucleic acid with saidTHAP-family polypeptide;

(e) isolating a complex between said at least one amplified nucleic acidand said THAP-family polypeptide;

(f) repeating steps (c), (d) and (e) a plurality of times;

(g) determining the sequence of said nucleic acid in said complex.

119. A method of identifying a compound which inhibits the ability of aTHAP-family polypeptide to bind to a nucleic acid comprising: incubatinga THAP-family polypeptide or a fragment thereof which recognizes abinding site in a nucleic acid with a nucleic acid containing saidbinding site in the presence or absence of a test compound anddetermining whether the level of binding of said THAP-family polypeptideto said nucleic acid in the presence of said test compound is less thanthe level of binding in the absence of said test compound.

120. A method of identifying a test compound that modulatesTHAP-mediated activities comprising:

-   -   contacting a THAP-family polypeptide or a biologically active        fragment thereof with a test compound, wherein said THAP-family        polypeptide comprises an amino acid sequence having at least 30%        amino acid identity to an amino acid sequence of SEQ ID NO: 1;        and    -   determining whether said test compound selectively modulates the        activity of said THAP-family polypeptide or biologically active        fragment thereof, wherein a determination that said test        compound selectively modulates the activity of said polypeptide        indicates that said test compound is a candidate modulator of        THAP-mediated activities.

121. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 1, or a biologicallyactive fragment thereof.

122. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 2, or a biologicallyactive fragment thereof.

123. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 3, or a biologicallyactive fragment thereof.

124. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 4, or a biologicallyactive fragment thereof.

125. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 5, or a biologicallyactive fragment thereof.

126. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 6, or a biologicallyactive fragment thereof.

127. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 7, or a biologicallyactive fragment thereof.

128. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 8, or a biologicallyactive fragment thereof.

129. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 9, or a biologicallyactive fragment thereof.

130. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 10, or a biologicallyactive fragment thereof.

131. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 11, or a biologicallyactive fragment thereof.

132. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 12, or a biologicallyactive fragment thereof.

133. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 13, or a biologicallyactive fragment thereof.

134. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence of SEQ ID NO: 14 or a biologicallyactive fragments thereof.

135. The method of Paragraph 120, wherein the THAP-family polypeptidecomprises the amino acid sequence selected from the group consisting ofSEQ ID NOs: 15-114 or a biologically active fragments thereof.

136. The method of Paragraph 120, wherein said THAP-mediated activity isselected from the group consisting of interaction with a THAP-familytarget protein, binding to a nucleic acid, binding to PAR-4, binding toSLC, binding to PML, binding to a polypeptide found in PML-NBs,localization to PML-NBs, targeting a THAP-family target protein toPML-NBs, and inducing apoptosis

137. The method of Paragraph 136, wherein said THAP-mediated activity isbinding to PAR-4.

138. The method of Paragraph 136, wherein said THAP-mediated activity isbinding to SLC.

139. The method of Paragraph 136, wherein said THAP-mediated activity isinducing apoptosis.

140. The method of Paragraph 136, wherein said nucleic acid comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:140-159.

141. The method of Paragraph 120, wherein said amino acid identity isdetermined using an algorithm selected from the group consisting ofXBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST withthe default parameters of XBLAST, and BLAST with the defaul parametersof XBLAST.

142. An isolated or purified THAP domain polypeptide consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs: 1-2, amino acids 1-89 of SEQ ID NOs: 3-5, amino acids1-90 of SEQ ID NOs: 6-9, amino acids 1-92 of SEQ ID NO: 10, amino acids1-90 of SEQ ID NOs: 11-14 and homologs having at least 30% amino acididentity to any aforementioned sequence, wherein said polypeptide bindsto a nucleic acid.

143. The isolated or purified THAP domain polypeptide of Paragraph 142consisting essentially of SEQ ID NO: 1.

144. The isolated or purified THAP domain polypeptide of Paragraph 142,wherein said amino acid identity is determined using an algorithmselected from the group consisting of XBLAST with the parameters,score=50 and wordlength=3, Gapped BLAST with the default parameters ofXBLAST, and BLAST with the defaul parameters of XBLAST.

145. The isolated or purified THAP domain polypeptide of Paragraph 142,wherein said nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 140-159.

146. An isolated or purified nucleic acid which encodes the THAP domainpolypeptide of Paragraph 142 or a complement thereof.

147. An isolated or purified PAR4-binding domain polypeptide consistingessentially of an amino acid sequence selected from the group consistingof amino acids 143-192 of SEQ ID NO: 3, amino acids 132-181 of SEQ IDNO: 4, amino acids 186-234 of SEQ ID NO: 5, SEQ ID NO: 15 and homologshaving at least 30% amino acid identity to any aforementioned sequence,wherein said polypeptide binds to PAR4.

148. The isolated or purified PAR4-binding domain of Paragraph 147consisting essentially of SEQ ID NO: 15.

149. The isolated or purified PAR4-binding domain of Paragraph 147consisting essentially of amino acids 143-193 of SEQ ID NO: 3.

150. The isolated or purified PAR4-binding domain of Paragraph 147consisting essentially of amino acids 132-181 of SEQ ID NO: 4.

151. The isolated or purified PAR4-binding domain of Paragraph 147consisting essentially of amino acids 186-234 of SEQ ID NO: 5.

152. The isolated or purified PAR4-binding domain polypeptide ofParagraph 147, wherein said amino acid identity is determined using analgorithm selected from the group consisting of XBLAST with theparameters, score=50 and wordlength=3, Gapped BLAST with the defaultparameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

153. An isolated or purified nucleic acid which encodes the PAR4-bindingdomain polypeptide of Paragraph 147 or a complement thereof.

154. An isolated or purified SLC-binding domain polypeptide consistingessentially of an amino acid sequence selected from the group consistingof amino acids 143-213 of SEQ ID NO: 3 and homologs thereof having atleast 30% amino acid identity, wherein said polypeptide binds to SLC.

155. The isolated or purified SLC-binding domain polypeptide ofParagraph 15:4, wherein said amino acid identity is determined using analgorithm selected from the group consisting of XBLAST with theparameters, score=50 and wordlength=3, Gapped BLAST with the defaultparameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

156. An isolated or purified nucleic acid which encodes the SLC-bindingdomain polypeptide of Paragraph 154 or a complement thereof.

157. A fusion protein comprising an Fc region of an immunoglobulin fusedto a polypeptide comprising an amino acid sequence selected from thegroup consisting of amino acids 143-213 of SEQ ID NO: 3 and homologsthereof having at least 30% amino acid identity.

158. An oligomeric THAP protein comprising a plurality of THAPpolypeptides, wherein each THAP polypeptide comprises an amino acidsequence selected from the group consisting of amino acid 143-213 of SEQID NO: 3 and homologs thereof having at least 30% amino acid identity.

159. A medicament comprising an effective amount of a THAP1 polypeptideor an SLC-binding fragment thereof, together with a pharmaceuticallyacceptable carrier.

160. An isolated or purified THAP dimerization domain polypeptideconsisting essentially of an amino acid sequence selected from the groupconsisting of amino acids 143 and 192 of SEQ ID NO: 3 and homologsthereof having at least 30% amino acid identity, wherein saidpolypeptide binds to a THAP-family polypeptide.

161. The isolated or purified THAP dimerization domain polypeptide ofParagraph 160, wherein said amino acid identity is determined using analgorithm selected from the group consisting of XBLAST with theparameters, score=50 and wordlength=3, Gapped BLAST with the defaultparameters of XBLAST, and BLAST with the defaul parameters of XBLAST.

162. An isolated or purified nucleic acid which encodes the THAPdimerization domain polypeptide of Paragraph 160 or a complementthereof.

163. An expression vector comprising a promoter operably linked to anucleic acid having a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 160-175 and portions thereof comprising atleast 18 consecutive nucleotides.

164. The expression vector of Paragraph 163, wherein said promoter is apromoter which is not operably linked to said nucleic acid selected fromthe group consisting of SEQ ID NOs.: 160-175 in a naturally occurringgenome.

165. A host cell comprising the expression vector of Paragraph 163.

166. An expression vector comprising a promoter operably linked to anucleic acid encoding a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-114 and portionsthereof comprising at least 18 consecutive nucleotides.

167. The expression vector of Paragraph 166, wherein said promoter is apromoter which is not operably linked to said nucleic acid selected fromthe group consisting of SEQ ID NOs.: 160-175 in a naturally occurringgenome.

168. A host cell comprising the expression vector of Paragraph 166.

169. A method of identifying a candidate inhibitor of a THAP-familypolypeptide, a candidate inhibitor of apoptosis, or a candidate compoundfor the treatment of a cell proliferative disorder, said methodcomprising:

-   -   contacting a THAP-family polypeptide comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 1-114        or a fragment comprising a span of at least 6 contiguous amino        acids of a polypeptide comprising an amino acid sequence        selected from the group consisting of SEQ ID NOs: 1-114 with a        test compound; and    -   determining whether said compound selectively binds to said        polypeptide, wherein a determination that said compound        selectively binds to said polypeptide indicates that said        compound is a candidate inhibitor of a THAP-family polypeptide,        a candidate inhibitor of apoptosis, or a candidate compound for        the treatment of a cell proliferative disorder.

170. A method of identifying a candidate inhibitor of apoptosis, acandidate compound for the treatment of a cell proliferative disorder,or a candidate inhibitor of a THAP-family polypeptide of SEQ ID NOs:1-114 or a fragment comprising a span of at least 6 contiguous aminoacids of a polypeptide according to SEQ ID NOs: 1-114, said methodcomprising:

-   -   contacting said THAP-family polypeptide with a test compound;        and    -   determining whether said compound selectively inhibits at least        one biological activity selected from the group consisting of        interaction with a THAP-family target protein, binding to a        nucleic acid sequence, binding to PAR-4, binding to SLC, binding        to PML, binding to a polypeptide found in PML-NBs, localization        to PML-NBs, targeting a THAP-family target protein to PML-NBs,        and inducing apoptosis, wherein a determination that said        compound selectively inhibits said at least one biological        activity of said polypeptide indicates that said compound is a        candidate inhibitor of a THAP-family polypeptide, a candidate        inhibitor of apoptosis, or a candidate compound for the        treatment of a cell proliferative disorder.

171. A method of identifying a candidate inhibitor of apoptosis, acandidate compound for the treatment of a cell proliferative disorder,or a candidate inhibitor of a THAP-family polypeptide of SEQ ID NOs:1-114 or a fragment comprising a span of at least 6 contiguous aminoacids of a polypeptide according to SEQ ID NOs: 1-114, said methodcomprising:

-   -   contacting a cell comprising said THAP-family polypeptide with a        test compound; and    -   determining whether said compound selectively inhibits at least        one biological activity selected from the group consisting of        interaction with a THAP-family target protein, binding to a        nucleic acid sequence, binding to PAR-4, binding to SLC, binding        to PML, binding to a polypeptide found in PML-NBs, localization        to PML-NBs, targeting a THAP-family target protein to PML-NBs,        and inducing apoptosis, wherein a determination that said        compound selectively inhibits said at least one biological        activity of said polypeptide indicates that said compound is a        candidate inhibitor of a THAP-family polypeptide, a candidate        inhibitor of apoptosis, or a candidate compound for the        treatment of a cell proliferative disorder.

172. A method of identifying a candidate modulator of THAP-familyactivity, said method comprising:

-   -   providing a THAP-family polypeptide of SEQ ID NOs: 1-114 or, a        fragment comprising a span of at least 6 contiguous amino acids        of a polypeptide according to SEQ ID NOs: 1-114; and    -   providing a THAP-family target polypeptide or a fragment        thereof; and    -   determining whether a test compound selectively modulates the        ability of said THAP-family polypeptide to bind to said        THAP-family target polypeptide, wherein a determination that        said test compound selectively modulates the ability of said        THAP-family polypeptide to bind to said THAP-family target        polypeptide indicates that said compound is a candidate        modulator of THAP-family activity.

173. The method of Paragraph 172, wherein said THAP-family polypeptideis provided by a first expression vector comprising a nucleic acidencoding a THAP-family polypeptide of SEQ ID NOs: 1-114 or, a fragmentcomprising a contiguous span of at least 6 contiguous amino acids of apolypeptide according to SEQ ID NOs: 1-114, and wherein said THAP-familytarget polypeptide is provided by a second expression vector comprisinga nucleic acid encoding a THAP-family target polypeptide, or a fragmentthereof.

174. The method of Paragraph 172, wherein said THAP-family activity isapoptosis activity.

175. The method of Paragraph 172, wherein said THAP-family targetprotein is PAR-4.

176. The method of Paragraph 172, wherein said THAP-family polypeptideis a THAP-1, THAP-2 or THAP-3 protein and said THAP-family targetprotein is PAR-4.

177. The method of Paragraph 172, wherein said THAP-family targetprotein is SLC.

178. A method of modulating apoptosis in a cell comprising modulatingthe activity of a THAP-family protein.

179. The method of Paragraph 178, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs: 1-114.

180. The method of Paragraph 178, wherein modulating the activity of aTHAP-family protein comprises modulating the interaction of aTHAP-family protein and a THAP-family target protein.

181. The method of Paragraph 178, wherein modulating the activity of aTHAP-family protein comprises modulating the interaction of aTHAP-family protein and a PAR4 protein.

182. A method of identifying a candidate activator of a THAP-familypolypeptide, a candidate activator of apoptosis, or a candidate compoundfor the treatment of a cell proliferative disorder, said methodcomprising:

-   -   contacting a THAP-family polypeptide comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 1-98        or a fragment comprising a span of at least 6 contiguous amino        acids of a polypeptide comprising an amino acid sequence        selected from the group consisting of SEQ ID NOs: 1-98 with a        test compound; and    -   determining whether said compound selectively binds to said        polypeptide, wherein a determination that said compound        selectively binds to said polypeptide indicates that said        compound is a candidate activator of a THAP-family polypeptide,        a candidate activator of apoptosis, or a candidate compound for        the treatment of a cell proliferative disorder.

183. A method of identifying a candidate activator of apoptosis, acandidate compound for the treatment of a cell proliferative disorder,or a candidate activator of a THAP-family polypeptide of SEQ ID NOs:1-98 or a fragment comprising a span of at least 6 contiguous aminoacids of a polypeptide according to SEQ ID NOs: 1-98, said methodcomprising:

-   -   contacting said THAP-family polypeptide with a test compound;        and    -   determining whether said compound selectively activates at least        one biological activity selected from the group consisting of        interaction with a THAP-family target protein, binding to a        nucleic acid sequence, binding to PAR-4, binding to SLC, binding        to PML, binding to a polypeptide found in PML-NBs, localization        to PML-NBs, targeting a THAP-family target protein to PML-NBs,        and inducing apoptosis, wherein a determination that said        compound selectively activates said at least one biological        activity of said polypeptide indicates that said compound is a        candidate activator of a THAP-family polypeptide, a candidate        activator of apoptosis, or a candidate compound for the        treatment of a cell proliferative disorder.

184. A method of identifying a candidate activator of apoptosis, acandidate compound for the treatment of a cell proliferative disorder,or a candidate activator of a THAP-family polypeptide of SEQ ID NOs: 1to 98 or a fragment comprising a span of at least 6 contiguous aminoacids of a polypeptide according to SEQ ID NOs: 1-98, said methodcomprising:

-   -   contacting a cell comprising said THAP-family polypeptide with a        test compound; and    -   determining whether said compound selectively activates at least        one biological activity selected from the group consisting of        interaction with a THAP-family target protein, binding to a        nucleic acid sequence, binding to PAR-4, binding to SLC, binding        to PML, binding to a polypeptide found in PML-NBs, localization        to PML-NBs, targeting a THAP-family target protein to PML-NBs,        and inducing apoptosis, wherein a determination that said        compound selectively activates said at least one biological        activity of said polypeptide indicates that said compound is a        candidate activator of a THAP-family polypeptide, a candidate        activator of apoptosis, or a candidate compound for the        treatment of a cell proliferative disorder.

185. A method of ameliorating a condition associated with the activityof SLC in an individual comprising administering a polypeptidecomprising the SLC binding domain of a THAP-family protein to saidindividual.

186. The method of Paragraph 185, wherein said polypeptide comprises afusion protein comprising an Fe region of an immunoglobulin fused to apolypeptide comprising an amino acid sequence selected from the groupconsisting of amino acids 143-213 of SEQ ID NO: 3 and homologs thereofhaving at least 30% amino acid identity.

187. The method of Paragraph 185, wherein said polypeptide comprises anoligomeric THAP protein comprising a plurality of THAP polypeptides,wherein each THAP polypeptide comprises an amino acid sequence selectedfrom the group consisting of amino acid 143-213 of SEQ ID NO: 3 andhomologs thereof having at least 30% amino acid identity.

188. A method of modulating angiogenesis in an individual comprisingmodulating the activity of a THAP-family protein in said individual.

189. The method of Paragraph 188, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs: 1-114.

190. The method of Paragraph 188, wherein said modulation is inhibition.

191. The method of Paragraph 188, wherein said modulation is induction.

192. A method of reducing cell death in an individual comprisinginhibiting the activity of a THAP-family protein in said individual.

193. The method of Paragraph 192, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs: 1-114.

194. The method according to Paragraph 192, wherein the activity of saidTHAP-family protein is inhibited in the CNS.

195. A method of reducing inflammation or an inflammatory disorder in anindividual comprising modulating the activity of a THAP-family proteinin said individual.

196. The method of Paragraph 195, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs: 1-114.

197. A method of reducing the extent of cancer in an individualcomprising modulating the activity of a THAP-family protein in saidindividual.

198. The method of Paragraph 197, wherein said THAP-family protein isselected from the group consisting of SEQ ID NOs: 1-114.

199. The method of Paragraph 197, wherein increasing the activity ofsaid THAP family protein induces apoptosis, inhibits cell division,inhibits metastatic potential, reduces tumor burden, increasessensitivity to chemotherapy or radiotherapy, kills a cancer cell,inhibits the growth of a cancer cell, kills an endothelial cell,inhibits the growth of an endothelial cell, inhibits angiogenesis, orinduces tumor regression.

200. A method of forming a complex, said method comprising:

-   -   contacting a chemokine with a chemokine-binding agent comprising        a polypeptide selected from the group consisting of THAP-1, a        polypeptide having at least 30% amino acid identity to THAP-1, a        chemokine-binding domain of THAP-1 and a polypeptide having at        least 30% amino acid identity to a chemokine-binding domain of        THAP-1, wherein said chemokine and said chemokine binding agent        form a complex.

201. The method of Paragraph 200, wherein said amino acid identity isdetermined using an algorithm selected from the group consisting ofXBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST withthe default parameters of XBLAST, and BLAST with the defaul parametersof XBLAST.

202. The method of Paragraph 200, wherein said polypeptide is fused toan Fc region of an immunoglobulin.

203. The method of Paragraph 200, wherein said polypeptide comprises aTHAP dimerization domain.

204. The method of Paragraph 203, wherein said THAP dimerization domaininteracts with one or more THAP dimerization domains to form a THAPoligomer.

205. The method of Paragraph 200, wherein said polypeptide is arecombinant polypeptide.

206. The method of Paragraph 200, wherein said chemokine is selectedfrom the group consisting of SLC, CCL19, CCL5, CXCL9 and CXCL10.

207. The method of Paragraph 200, wherein said chemokine is selectedfrom the group consisting of SLC, CCL19 and CXCL9.

208. The method of Paragraph 200, wherein said polypeptide comprisesTHAP-1.

209. The method of Paragraph 208, wherein said THAP-1 comprises theamino acid sequence of SEQ ID NO: 3.

210. The method of Paragraph 200, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to THAP-1.

211. The method of Paragraph 200, wherein said polypeptide comprises achemokine-binding domain of THAP-1.

212. The method of Paragraph 211, wherein said chemokine-binding domainof THAP-1 comprises the amino acid sequence of amino acids 143-213 ofSEQ ID NO: 3.

213. The method of Paragraph 200, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to achemokine-binding domain of THAP-1.

214. A method of inhibiting the activity of a chemokine, said methodcomprising contacting a chemokine with an effective amount of an agentcomprising a polypeptide selected from the group consisting of THAP-1, apolypeptide having at least 30% amino acid identity to THAP-1, achemokine-binding domain of THAP-1 and a polypeptide having at least 30%amino acid identity to a chemokine-binding domain of THAP-1, wherein theactivity of said chemokine is inhibited.

215. The method of Paragraph 214, wherein said amino acid identity isdetermined using an algorithm selected from the group consisting ofXBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST withthe default parameters of XBLAST, and BLAST with the defaul parametersof XBLAST.

216. The method of Paragraph 214, wherein said polypeptide is fused toan Fc region of an immunoglobulin.

217. The method of Paragraph 214, wherein said polypeptide comprises aTHAP dimerization domain.

218. The method of Paragraph 217, wherein said THAP dimerization domaininteracts with one or more THAP dimerization domains to form a THAPoligomer.

219. The method of Paragraph 214, wherein said polypeptide is arecombinant polypeptide.

220. The method of Paragraph 214, wherein said polypeptide binds to achemokine selected from the group consisting of SLC, CCL19, CCL5, CXCL9and CXCL10.

221. The method of Paragraph 214, wherein said polypeptide binds to achemokine selected from the group consisting of SLC, CCL19 and CXCL9.

222. The method of Paragraph 214, wherein said polypeptide comprisesTHAP-1.

223. The method of Paragraph 222, wherein said THAP-1 comprises theamino acid sequence of SEQ ID NO: 3.

224. The method of Paragraph 214, wherein said polypeptide comprises apolypeptide having at least 30% amino acid. identity to THAP-1.

225. The method of Paragraph 214, wherein said polypeptide comprises achemokine-binding domain of THAP-1.

226. The method of Paragraph 225, wherein said chemokine-binding domainof THAP-1 comprises the amino acid sequence of amino acids 143-213 ofSEQ ID NO: 3.

227. The method of Paragraph 214, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to achemokine-binding domain of THAP-1.

228. A method of reducing inflammation comprising administering aneffective amount of a chemokine binding agent to a subject afflictedwith an inflammatory condition, wherein said chemokine-binding agentcomprises a polypeptide selected from the group consisting of THAP-1, apolypeptide having at least 30% amino acid identity to THAP-1, achemokine-binding domain of THAP-1 and a polypeptide having at least 30%amino acid identity to a chemokine-binding domain of THAP-1.

229. The method of Paragraph 228, wherein said amino acid identity isdetermined using an algorithm selected from the group consisting ofXBLAST with the parameters, score=50 and wordlength=3, Gapped BLAST withthe default parameters of XBLAST, and BLAST with the defaul parametersof XBLAST.

230. The method of Paragraph 228, wherein said polypeptide is fused toan Fc region of an immunoglobulin.

231. The method of Paragraph 228, wherein said polypeptide comprises aTHAP dimerization domain.

232. The method of Paragraph 231, wherein said THAP dimerization domaininteracts with one or more THAP dimerization domains to form a THAPoligomer.

233. The method of Paragraph 228, wherein said polypeptide is arecombinant polypeptide.

234. The method of Paragraph 228, wherein said polypeptide binds to achemokine selected from the group consisting of SLC, CCL19, CCL5, CXCL9and CXCL10.

235. The method of Paragraph 228, wherein said polypeptide binds to achemokine selected from the group consisting of SLC, CCL19 and CXCL9.

236. The method of Paragraph 228, wherein said polypeptide comprisesTHAP-1.

237. The method of Paragraph 236, wherein said THAP-1 comprises theamino acid sequence of SEQ ID NO: 3.

238. The method of Paragraph 228, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to THAP-1.

239. The method of Paragraph 228, wherein said polypeptide comprises achemokine-binding domain of THAP-1.

240. The method of Paragraph 239, wherein said chemokine-binding domainof THAP-1 comprises the amino acid sequence of amino acids 143-213 ofSEQ ID NO: 3.

241. The method of Paragraph 228, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to achemokine-binding domain of THAP-1.

242. A method of reducing one or more symptoms associated with aninflammatory disease, said method comprising administering to a subjectafflicted with said inflammatory disease a therapeutically effectiveamount of an agent which reduces or eliminates the activity of one ormore chemokines, wherein said agent comprises a polypeptide selectedfrom the group consisting of THAP-1, a polypeptide having at least 30%amino acid identity to THAP-1, a chemokine-binding domain of THAP-1 anda polypeptide having at least 30% amino acid identity to achemokine-binding domain of THAP-1.

243. The method of Paragraph 242, wherein said polypeptide is fused toan Fc region of an immunoglobulin.

244. The method of Paragraph 242, wherein said polypeptide comprises aTHAP dimerization domain.

245. The method of Paragraph 244, wherein said THAP dimerization domaininteracts with one or more THAP dimerization domains to form a THAPoligomer.

246. The method of Paragraph 242, wherein said polypeptide is arecombinant polypeptide.

247. The method of Paragraph 242, wherein said polypeptide binds to achemokine selected from the group consisting of SLC, CCL19, CCL5, CXCL9and CXCL 10.

248. The method of Paragraph 242, wherein said polypeptide binds to achemokine selected from the group consisting of SLC, CCL19 and CXCL9.

249. The method of Paragraph 242, wherein said polypeptide comprisesTHAP-1.

250. The method of Paragraph 249, wherein said THAP-1 comprises theamino acid sequence of SEQ ID NO: 3.

251. The method of Paragraph 242, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to THAP-1.

252. The method of Paragraph 242, wherein said polypeptide comprises achemokine-binding domain of THAP-1.

253. The method of Paragraph 252, wherein said chemokine-binding domainof THAP-1 comprises the amino acid sequence of amino acids 143-213 ofSEQ ID NO: 3.

254. The method of Paragraph 242, wherein said polypeptide comprises apolypeptide having at least 30% amino acid identity to achemokine-binding domain of THAP-1.

255. The method of Paragraph 242, wherein said inflammatory disease isarthritis.

256. The method of Paragraph 242, wherein said inflammatory disease isinflammatory bowel disease.

257. A method of detecting a chemokine, said method comprising:

-   -   contacting a chemokine with a chemokine-binding agent comprising        a polypeptide selected from the group consisting of THAP-1, a        polypeptide having at least 30% amino acid identity to THAP-1, a        chemokine-binding domain of THAP-1 and a polypeptide having at        least 30% amino acid identity to a chemokine-binding domain of        THAP-1; and    -   detecting chemokine-binding agent bound to said chemokine.

258. The method of Paragraph 257, wherein chemokine is selected from thegroup consisting of SLC, CCL19, CCL5, CXCL9 and CXCL10.

259. The method of Paragraph 257, wherein said chemokine is selectedfrom the group consisting of SLC, CCL19 and CXCL9.

260. A detection system comprising a chemokine-binding agent comprisinga polypeptide selected from the group consisting of THAP-1, apolypeptide having at least 30% amino acid identity to THAP-1, achemokine-binding domain of THAP-1 and a polypeptide having at least 30%amino acid identity to a chemokine-binding domain of THAP-1, whereinsaid chemokine-binding agent is coupled to a solid support.

261. The detection system of Paragraph 260, wherein said polypeptidecomprises THAP-1.

262. The detection system of Paragraph 261, wherein said THAP-1comprises the amino acid sequence of SEQ ID NO: 3.

263. The detection system of Paragraph 260, wherein said polypeptidecomprises a polypeptide having at least 30% amino acid identity toTHAP-1.

264. The detection system of Paragraph 260, wherein said polypeptidecomprises a chemokine-binding domain of THAP-1.

265. The detection system of Paragraph 264, wherein saidchemokine-binding domain of THAP-1 comprises the amino acid sequence ofamino acids 143-213 of SEQ ID NO: 3.

266. The detection system of Paragraph 260, wherein said polypeptidecomprises a polypeptide having at least 30% amino acid identity to achemokine-binding domain of THAP-1.

267. A pharmaceutical composition comprising a chemokine-binding agentin a pharaceutically acceptable carrier, wherein said chemokine-bindingagent comprises a polypeptide selected from the group consisting ofTHAP-1, a polypeptide having at least 30% amino acid identity to THAP-1,a chemokine-binding domain of THAP-1 and a polypeptide having at least30% amino acid identity to a chemokine-binding domain of THAP-1.

268. The pharmaceutical composition of Paragraph 267, wherein said aminoacid identity is determined using an algorithm selected from the groupconsisting of XBLAST with the parameters, score=50 and wordlength=3,Gapped BLAST with the default parameters of XBLAST, and BLAST with thedefaul parameters of XBLAST.

269. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide is fused to an Fc region of an immunoglobulin.

270. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide comprises a THAP dimerization domain.

271. The pharmaceutical composition of Paragraph 270, wherein said THAPdimerization domain interacts with one or more THAP dimerization domainsto form a THAP oligomer.

272. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide binds to a chemokine selected from the group consisting ofSLC, CCL19, CCL5, CXCL9 and CXCL10.

273. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide binds to a chemokine selected from the group consisting ofSLC, CCL19 and CXCL9.

274. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide comprises THAP-1.

275. The pharmaceutical composition of Paragraph 274, wherein saidTHAP-1 comprises the amino acid sequence of SEQ ID NO: 3.

276. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide comprises a polypeptide having at least 30% amino acididentity to THAP-1.

277. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide comprises a chemokine-binding domain of THAP-1.

278. The pharmaceutical composition of Paragraph 277, wherein saidchemokine-binding domain of THAP-1 comprises the amino acid sequence ofamino acids 143-213 of SEQ ID NO: 3.

279. The pharmaceutical composition of Paragraph 267, wherein saidpolypeptide comprises a polypeptide having at least 30% amino acididentity to a chemokine-binding domain of THAP-1.

280. A device for administering an agent, said device comprising acontainer that contains therein a chemokine-binding agent in apharmaceutically acceptable carrier, wherein said chemokine-bindingagent comprises a polypeptide selected from the group consisting ofTHAP-1, a polypeptide having at least 30% amino acid identity to THAP-1,a chemokine-binding domain of THAP-1 and a polypeptide having at least30% amino acid identity to a chemokine-binding domain of THAP-1.

281. The device according to Paragraph 280, wherein said container is asyringe.

282. The device according to Paragraph 280, wherein said container is apatch for transdermal administration.

283. The device according to Paragraph 280, wherein said container ispressurized canister.

284. A kit comprising:

-   -   a chemokine-binding agent comprising a polypeptide selected from        the group consisting of THAP-1, a polypeptide having at least        30% amino acid identity to THAP-1, a chemokine-binding domain of        THAP-1 and a polypeptide having at least 30% amino acid identity        to a chemokine-binding domain of THAP-1; and    -   instructions for using said chemokine-binding agent for        detecting or inhibiting chemokines.

285. The kit of Paragraph 284, wherein said chemokine is selected fromthe group consisiting of SLC, CCL19, CCL5, CXCL9 and CXCL10.

286. An isolated or purified chemokine-binding domain consistingessentially of a portion of SEQ ID NO: 3 that binds to a chemokine.

287. The isolated or purified chemokine-binding domain of Paragraph 286,wherein said chemokine is CCL19.

288. The isolated or purified chemokine-binding domain of Paragraph 286,wherein said chemokine is CCL5.

289. The isolated or purified chemokine-binding domain of Paragraph 286,wherein said chemokine is CXCL9.

290. The isolated or purified chemokine-binding domain of Paragraph 286,wherein said chemokine is CXCL 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an amino acid sequence alignment of human THAP1(hTHAP1) (SEQ ID NO: 3) and mouse THAP1 (mTHAP1) (SEQ ID NO: 99)orthologous polypeptides. Identical amino acid residues are indicatedwith an asterisk.

FIG. 1B depicts the primary structure of the human THAP1 polypeptide.Positions of the THAP domain, the proline-rich region (PRO) and thebipartite nuclear localization sequence (NLS) are indicated.

FIG. 2 depicts the results of a Northern Blot analysis of THAP1 mRNAexpression in 12 human tissues. Each lane contains 2 μg of poly A⁺ RNAisolated from the indicated human tissues. The blot was hybridized,under high-stringency conditions, with a ³²P-labeled THAP1 cDNA probe,and exposed at −70° C. for 72 hours.

FIG. 3A illustrates the interaction between THAP1 and PAR4 in a yeasttwo-hybrid system. In particular, THAP1 binds to wild-type Par4 (Par4)and the leucine zipper-containing Par4 death domain (Par4DD) (aminoacids 250-342 of PAR4) but not a Par4 deletion mutant lacking the deathdomain (PAR4Δ) (amino acids 1-276 of PAR4). A (+) indicates bindingwhereas a (−) indicated lack of binding.

FIG. 3B shows the binding of in vitro translated,³⁵S-methionine-labeledTHAP1 to a GST-Par4DD polypeptide fusion. Par4DD was expressed as a GSTfusion protein then purified on an affinity matrix of glutathionesepharose. GST served as negative control. The input represents 1/10 ofthe material used in the binding assay.

FIG. 4A illustrates the interaction between PAR4 and several THAP1deletion mutants both in vitro and in vivo. Each THAP1 deletion mutantwas tested for binding to either PAR or PAR4DD in a yeast two hybridsystem (two hybrid bait), to PAR4DD in GST pull down assays (in vitro)and to myc-Par4DD in primary human endothelial cells (in vivo). A (+)indicates binding whereas a (−) indicated lack of binding.

FIG. 4B shows the binding of several in vitro translated,³⁵S-methionine-labeled THAP1 deletion mutants to a GST-Par4DDpolypeptide fusion. Par4DD was expressed as a GST fusion protein thenpurified on an affinity matrix of glutathione sepharose. GST served asnegative control. The input represents 1/10 of the material used in thebinding assay.

FIG. 5A depicts an amino acid sequence alignment of the Par4 bindingdomain of human THAP1 (SEQ ID NO: 117) and mouse THAP1 (SEQ ID NO: 116)orthologues with that of mouse ZIP kinase (SEQ ID NO: 115), another Par4binding partner. An arginine-rich consensus Par4 binding site (SEQ IDNO: 15), derived from this alignment, is also indicated.

FIG. 5B shows the primary structure of the THAP1 wild-type polypeptideand two THAP1 mutants (THAP1Δ(QRCRR) and THAP1 RR/AA). THAP1Δ(QRCRR) isa deletion mutant having a deletion of amino acids at positions 168-172of THAP1 (SEQ ID NO: 3) whereas THAP RR/AA is a mutant having the twoarginines located at amino acid positions 171 and 172 to THAP1 (SEQ IDNO: 3) replaced with alanines. Results obtained, in yeast two-hybridsystem with Par4 and Par4DD baits (two hybrid bait), in GST pull downassays with GST-Par4DD (in vitro) and in the in vivo interaction testwith myc-Par4DD in primary human endothelial cells (in vivo) aresummarized.

FIG. 6A is a graph which compares apoptosis levels in cells transfectedwith GFP-APSK1, GFP-Par4 or GFP-THAP1 expression vectors. Apoptosis wasquantified by DAPI staining of apoptotic nuclei, 24 h afterserum-withdrawal. Values are the means of three independent experiments.

FIG. 6B is a graph which compares apoptosis levels in cells transfectedwith GFP-APSK1 or GFP-THAP1 expression vectors. Apoptosis was quantifiedby DAPI staining of apoptotic nuclei, 24 h after addition of TNFα.Values are the means of three independent experiments.

FIG. 7A shows the binding of in vitro translated ³⁵S-methionine labeledTHAP1 (wt) or THAP1ΔTHAP (Δ) to a GST-Par4DD polypeptide fusion. Par4DDwas expressed as a GST fusion protein then purified on an affinitymatrix of glutathione sepharose. GST served as negative control. Theinput represents 1/10 of the material used in the binding assay.

FIG. 7B is a graph which compares the proapoptotic activity of THAP1with a THAP1 mutant having its THAP domain (amino acids 1-90 of SEQ IDNO: 3) deleted. The percentage of apoptotic cells in mouse 3T3fibroblasts overexpressing GFP-APSK1 (control), GFP-THAP1 (THAP1) orGFP-THAP1ΔTHAP (THAP1ΔTHAP) was determined by counting apoptotic nucleiafter DAPI staining. Values are the means of three independentexperiments.

FIG. 8 depicts the primary structure of twelve human THAP proteins. TheTHAP domain (colored grey) is located at the amino-terminus of each ofthe twelve human THAP proteins. The black box in THAP1, THAP2 and THAP3indicates a nuclear localization sequence, rich in basic residues, thatis conserved in the three proteins. The number of amino-acids in eachTHAP protein is indicated; (*) indicates the protein is not full length.

FIG. 9A depicts an amino acid sequence alignment of the THAP domain ofhuman THAP1 (hTHAP1, SEQ ID NO: 123) with the DNA binding domain ofdrosophila melanogaster P-element transposase (dmTransposase, SEQ ID NO:124). Identical residues are boxed in black and conserved residues ingrey. A THAP domain consensus sequence (SEQ ID NO: 125) is also shown.

FIG. 9B depicts an amino acid sequence alignment of the THAP domains oftwelve members of the human THAP family (hTHAP1, SEQ ID NO: 126; hTHAP2,SEQ ID NO: 131; hTHAP3, SEQ ID NO: 127; hTHAP4, SEQ ID NO: 130; hTHAP5,SEQ ID NO: 128; hTHAP6, SEQ ID NO: 135; hTHAP7, SEQ ID NO: 133; hTHAP8,SEQ ID NO: 129; hTHAP9, SEQ ID NO: 134; hTHAP10, SEQ ID NO: 137;hTHAP11, SEQ ID NO: 136; hTHAP0, SEQ ID NO: 132) with the DNA bindingdomain of Drosophila melanogaster P-element transposase (dmTransposae,SEQ ID NO: 138). Residues conserved among at least seven of the thirteensequences are boxed. Black boxes indicate identical residues whereasboxes shaded in grey show similar amino acids. Dashed lines representgaps introduced to align sequences. A THAP domain consensus sequence(SEQ ID NO: 139) is also shown.

FIG. 9C depicts an amino acid sequence alignment of 95 distinct THAPdomain sequences, including hTHAP1 through hTHAP11 and hTHAP0 (SEQ IDNOs: 3-14, listed sequentially beginning from the top), with 83 THAPdomains from other species (SEQ ID NOs: 16-98, listed sequentiallybeginning at the sequence denoted sTHAP1 and ending at the sequencedenoted ceNP_(—)498747.1), which were identified by searching GenBankgenomic and EST databases with the human THAP sequences. Residuesconserved among at least 50% of the sequences are boxed. Black boxesindicate identical residues whereas boxes shaded in grey show similaramino acids. Dashed lines represent gaps introduced to align sequences.The species are indicated: Homo sapiens (h); Sus scrofa (s); Bos taurus(b); Mus musculus (m); Rattus norvegicus (r); Gallus gallus (g); Xenopuslaevi (x); Danio rerio (z); Oryzias latipes (o); Drosophila melanogaster(dm); Anopheles gambiae (a); Bombyx mori (bm); Caenorhabditis.elegans(ce). A consensus sequence (SEQ ID NO: 2) is also shown. Amino acidsunderlined in the consensus sequence are residues which are conserved inall 95 THAP sequences.

FIG. 10A shows an amino acid sequence alignment of the human THAP1 (SEQID NO: 3), THAP2 (SEQ ID NO: 4) and THAP3 (SEQ ID NO: 5) proteinsequences. Residues conserved among at least two of the three sequencesare boxed. Black boxes indicate identical residues whereas boxes shadedin grey show similar amino acids. Dashed lines represent gaps introducedto align sequences. Regions corresponding to the THAP domain, thePAR4-binding domain, and the nuclear localization signal (NLS) are alsoindicated.

FIG. 10B shows the primary structure of human THAP1, THAP2 and THAP3 andresults of two-hybrid interactions between each THAP protein and Par4 orPar4 death domain (Par4DD) in the yeast two hybrid system.

FIG. 10C shows the binding of in vitro translated,³⁵S-methionine-labeledTHAP2 and THAP3 to a GST-Par4DD polypeptide fusion. Par4DD was expressedas a GST fusion protein then purified on an affinity matrix ofglutathione sepharose. GST served as negative control. The inputrepresents 1/10 of the material used in the binding assay.

FIG. 11A is a graph which compares apoptosis levels in cells transfectedwith GFP-APSK1, GFP-THAP2 or GFP-THAP3 expression vectors. Apoptosis wasquantified by DAPI staining of apoptotic nuclei, 24 h afterserum-withdrawal. Values are the means of two independent representativeexperiments.

FIG. 11B is a graph which compares apoptosis levels in cells transfectedwith GFP-APSK1, GFP-THAP2 or GFP-THAP3 expression vectors. Apoptosis wasquantified by DAPI staining of apoptotic nuclei, 24 h after additionalof TNFα. Values are the means of two independent representativeexperiments.

FIG. 12 illustrates the results obtained by screening several differentTHAP1 mutants in a yeast two-hybrid system with SLC/CCL21 bait. Theprimary structure of each THAP1 deletion mutant that was tested isshown. The 70 carboxy-terminal residues of THAP1 (amino acids 143-213)are sufficient for binding to chemokine SLC/CCL21.

FIG. 13 illustrates the interaction of THAP1 with wild type SLC/CCL21and a SLC/CCL21 mutant deleted of the basic carboxy-terminal extension(SLC/CCL21ΔCOOH). The interaction was analyzed both in yeast two-hybridsystem with THAP1 bait and in vitro using GST-pull down assays withGST-THAP1.

FIG. 14 depicts micrographs of the primary human endothelial cells weretransfected with the GFP-THAP0, 1, 2, 3, 6, 7, 8, 10, 11 (greenfluorescence) expression constructs. To reveal the nuclear localizationof the human THAP proteins, nuclei were counterstained with DAPI (blue).The bar equals 5 μm.

FIG. 15A is a threading-derived structural alignment between the THAPdomain of human THAP1 (THAP1) (amino acids 1-81 of SEQ ID NO: 3) and thethyroid receptor βDNA binding domain (NLLB) (SEQ ID NO: 121). The colorcoding is identical to that described in FIG. 15D.

FIG. 15B shows a model of the three-dimensional structure of the THAPdomain of human THAP1 based on its homology with the crystallographicstructure of thyroid receptor β. The color coding is identical to thatdescribed in FIG. 15D.

FIG. 15C shows a model of the three-dimensional structure of theDNA-binding domain of Drosophila transposase (DmTRP) based on itshomology with the crystallographic structure of the DNA-binding domainof the glucocorticoid receptor. The color coding is identical to thatdescribed in FIG. 15D.

FIG. 15D is a threading-derived structural alignment between theDrosophila melanogaster transposase DNA binding domain (DmTRP) (SEQ IDNO: 120) and the glucocorticoid receptor DNA binding domain (GLUA) (SEQID NO: 122). In accordance with the sequences and structures in FIGS.15A-15C, the color-coding is the following: brown indicates residues inα-helices; indigo indicates residues in β-strands; red denotes the eightconserved Cys residues in NLLB and GLUA or for the three Cys residuescommon to THAP1 and DmTRP; magenta indicates other Cys residues in THAP1or DmTRP; cyan denotes the residues involved in the hydrophobicinteractions networks colored in THAP1 or DmTRP.

FIG. 16A illustrates the results obtained by screening several differentTHAP1 mutants in a yeast two-hybrid system with THAP1 bait. The primarystructure of each THAP1 deletion mutant that was tested is shown. A (+)indicates binding whereas a (−) indicates no binding.

FIG. 16B shows the binding of several in vitro translated,³⁵S-methionine-labeled THAP1 deletion mutants to a GST-THAP1 polypeptidefusion. Wild-type THAP1 was expressed as a GST fusion protein thenpurified on an affinity matrix of glutathione sepharose. GST served asnegative control. The input represents 1/10 of the material used in thebinding assay.

FIG. 17A is an agarose gel showing two distinct THAP1 cDNA fragmentswere obtained by RT-PCR. Two distinct THAP1 cDNAs were ˜400 and 600nucleotides in length.

FIG. 17B shows that the 400 nucleotide fragment corresponds to analternatively spliced isoform of human THAP1 cDNA, lacking exon 2(nucleotides 273-468 of SEQ ID 160).

FIG. 17C is a Western blot which shows that the second isoform of humanTHAP1 (THAP1b) encodes a truncated THAP1 protein (THAP1 C3) lacking theamino-terminal THAP domain.

FIG. 18A shows a specific DNA binding site recognized by the THAP domainof human THAP1. The THAP domain recognizes GGGCAA or TGGCAA DNA targetsequences preferentially organized as direct repeats with 5 nucleotidespacing (DR-5). The consensus sequence 5′-GGGCAAnnnnnTGGCAA-3′ (SEQ IDNO: 149). The DR-5 consensus was generated by examination of 9 nucleicacids bound by THAP1 (SEQ ID NO: 140-148, beginning sequentially fromthe top).

FIG. 18B shows a second specific DNA binding site recognized by the THAPdomain of human THAP1. The THAP domain recognizes everted repeats with11 nucleotide spacing (ER-11) having a consensus sequence5′-TTGCCAnnnnnnnnnnGGGCAA-3′ (SEQ ID NO: 159). The ER-11 consensus wasgenerated by examination of 9 nucleic acids bound by THAP1 (SEQ ID NO:150-158, beginning sequentially from the top).

FIG. 19 shows that THAP1 interacts with both CC and CXC chemokines bothin vivo in a yeast two-hybrid system with THAP1 prey and in vitro usingGST-pull down assays with immobilized GST-THAP1. The cytokine IFNγ wasused as a negative control. Results are summarized as follows: +++indicates strong binding; ++ indicates intermediate binding; +/−indicates some binding; − indicates no binding; and ND indicates notdetermined.

FIG. 20A is an SDS-polyacrylamide gel showing the relative amounts ofchemokine and cytokine used in immobilized GST-THAP1 binding assays.

FIG. 20B is an SDS-polyacrylamide gel showing that neither the cytokine,IFNγ, nor any of the chemokines bound to immobilized GST alone.

FIG. 20 c is an SDS-polyacrylamide gel showing that chemokines, CXCL10,CXCL9 and CCL19, but not the cytokine IFNγ, bound to immobilizedGST-THAP1 fusions.

DETAILED DESCRIPTION OF THE INVENTION

THAP and PAR4 Biological Pathways

As mentioned above, the inventors have discovered a novel class ofproteins involved in apoptosis. Then, the inventors have also linked amember of this novel class to another (PAR4) apoptosis pathway, andfurther linked both of these pathways to PML-NBs. Moreover, theinventors have also linked both of these pathways to endothelial cells,providing a range of novel and potentially selective therapeutictreatments. In particular, it has been discovered that THAP1 (THanatos(death)-Associated-Protein-1) localizes to PML-NBs. Furthermore, twohybrid screening of an HEVEC cDNA library with the THAP1 bait lead tothe identification of a unique interacting partner, the pro-apoptoticprotein PAR4. PAR4 is also found to accumulate into PML-NBs. Targetingof the THAP-1/PAR4 complex to PML-NBs is mediated by PML. Similarly toPAR4, THAP1 has a pro-apoptotic activity. This activity includes a novelmotif in the amino-terminal part called THAP domain. Together theseresults define a novel PML-NBs pathway for apoptosis that involves theTHAP1/PAR4 pro-apoptotic complex.

THAP-Family Members, and Uses Thereof

The present invention includes polynucleotides encoding a family ofpro-apoptotic polypeptides THAP-0 to THAP11, and uses thereof for themodulation of apoptosis-related and other THAP-mediated activities.Included is THAP1, which forms a complex with the pro-apoptotic proteinPAR4 and localizes in discrete subnuclear domains known as PML nuclearbodies. Additionally, THAP-family polypeptides can be used to alter orotherwise modulate bioavailability of SLC/CCL21 (SLC).

The present invention also includes a novel protein motif, the THAPdomain, which is found in an 89 amino acid domain in the amino-terminalpart of THAP1 and which is involved in THAP1 pro-apoptotic activity. TheTHAP domain defines a novel family of proteins, the THAP-family, with atleast twelve distinct members in the human genome (THAP-0 to THAP11),which all contain a THAP domain in their amino-terminal part. Thepresent invention thus pertains to nucleic acid molecules, includinggenomic and in particular the complete cDNA sequences, encoding membersof the THAP-family, as well as with the corresponding translationproducts, nucleic acids encoding THAP domains, homologues thereof,nucleic acids encoding at least 10, 12, 15, 20, 25, 30, 40, 50, 100, 150or 200 consecutive amino acids, to the extent that said span isconsistent with the particular SEQ ID NO, of a sequence selected fromthe group consisting of SEQ ID NOs: 160-175.

THAP1 has been identified based on its expression in HEVs, specializedpostcapillary venules found in lymphoid tissues and nonlymphoid tissuesduring chronic inflammatory diseases that support a high level oflymphocyte extravasation from the blood. An important element in thecloning of the THAP1 cDNA from HEVECs was the development of protocolsfor obtaining HEVECs RNA, since HEVECs are not capable of maintainingtheir phenotype outside of their native environment for more than a fewhours. A protocol was developed where total RNA was obtained from HEVECsfreshly purified from human tonsils. Highly purified HEVECs wereobtained by a combination of mechanical and enzymatic procedures,immunomagnetic depletion and positive selection. Tonsils were mincedfinely with scissors on a steel screen, digested withcollagenase/dispase enzyme mix and unwanted contaminating cells werethen depleted using immunomagnetic depletion. HEVECs were then selectedby immunomagnetic positive selection with magnetic beads conjugated tothe HEV-specific antibody MECA-79. From these HEVEC that were 98%MECA-79-positive, 1 μg of total RNA was used to generate full lengthcDNAs for THAP1 cDNA cloning and RT-PCR analysis.

As used herein, the term “nucleic acids” and “nucleic acid molecule” isintended to include DNA molecules (e.g., cDNA or genomic DNA) and RNAmolecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. Throughout thepresent specification, the expression “nucleotide sequence” may beemployed to designate indifferently a polynucleotide or a nucleic acid.More precisely, the expression “nucleotide sequence” encompasses thenucleic material itself and is thus not restricted to the sequenceinformation (i.e. the succession of letters chosen among the four baseletters) that biochemically characterizes a specific DNA or RNAmolecule. Also, used interchangeably herein are terms “nucleic acids”,“oligonucleotides”, and “polynucleotides”.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated THAP-family nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleotide sequences which naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized. Anucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NOs: 160-175, aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all or aportion of the nucleic acid sequence of SEQ ID NOs: 160-175, as ahybridization probe, THAP-family nucleic acid molecules can be isolatedusing standard hybridization and cloning techniques (e.g., as describedin Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of e.g.SEQ ID NOs: 160-175, can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NOs: 160-175.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to THAP-family nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

As used herein, the term “hybridizes to” is intended to describeconditions for moderate stringency or high stringency hybridization,preferably where the hybridization and washing conditions permitnucleotide sequences at least 60% homologous to each other to remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 70%, more preferably at least about 80%, evenmore preferably at least about 85%, 90%, 95% or 98% homologous to eachother typically remain hybridized to each other. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are as follows: the hybridization step isrealized at 65° C. in the presence of 6×SSC buffer, 5× Denhardt'ssolution, 0,5% SDS and 100μg/ml of salmon sperm DNA. The hybridizationstep is followed by four washing steps:

two washings during 5 min, preferably at 65° C. in a 2×SSC and 0.1% SDSbuffer;

one washing during 30 min, preferably at 65° C. in a 2×SSC and 0.1% SDSbuffer,

one washing during 10 min, preferably at 65° C. in a 0.1×SSC and 0.1%SDS buffer,

these hybridization conditions being suitable for a nucleic acidmolecule of about 20 nucleotides in length. It will be appreciated thatthe hybridization conditions described above are to be adapted accordingto the length of the desired nucleic acid, following techniques wellknown to the one skilled in the art, for example be adapted according tothe teachings disclosed in Hames B. D. and Higgins S. J. (1985) NucleicAcid Hybridization: A Practical Approach. Hames and Higgins Ed., IRLPress, Oxford; and Current Protocols in Molecular Biolog (supra).Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to a sequence of SEQ ID NOs:160-175 corresponds to a naturally-occurring nucleic acid molecule. Asused herein, a “naturally-occurring” nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least 70%, 80%, 90%or 95% of the length of the reference sequence (e.g., when aligning asecond sequence to e.g. a THAP-1 amino acid sequence of SEQ ID NO: 3having 213 amino acid residues, at least 50, preferably at least 100,more preferably at least 200, amino acid residues are aligned or whenaligning a second sequence to the THAP-1 cDNA sequence of SEQ ID NO: 160having 2173 nucleotides or nucleotides 202-835 which encode the aminoacids of the THAP1 protein, preferably at least 100, preferably at least200, more preferably at least 300, even more preferably at least 400,and even more preferably at least 500, 600, at least 700, at least 800,at least 900, at least 1000, at least 1200, at least 1400, at least1600, at least 1800, or at least 2000 nucleotides are aligned. The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “identity” is equivalent to amino acid or nucleic acid “homology”).The percent homology between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %homology=number (#) of identical positions/total number (#) of positions100).

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77, thedisclosures of which are incorporated herein by reference in theirentireties. Such an algorithm is incorporated into the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to THAP-family nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to THAP-familyprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used (see,www.ncbi.nlm.nih.gov, the disclosures of which are incorporated hereinby reference in their entireties). Another preferred, non-limitingexample of a mathematical algorithim utilized for the comparison ofsequences is the algorithm of Myers and Miller, CABIOS (1989), thedisclosures of which are incorporated herein by reference in theirentireties. Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides, for example, polypeptides which include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theTHAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of a protein according to the invention (e.g. THAP familyor THAP domain polypeptide, or a biologically active fragment orhomologue thereof) in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a protein according to theinvention having less than about 30% (by dry weight) of protein otherthan the THAP-family protein (also referred to herein as a“contaminating protein”), more preferably less than about 20% of proteinother than the protein according to the invention, still more preferablyless than about 10% of protein other than the protein according to theinvention, and most preferably less than about 5% of protein other thanthe protein according to the invention. When the protein according tothe invention or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof inwhich the protein is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of a THAP-family protein havingless than about 30% (by dry weight) of chemical precursors ornon-THAP-family chemicals, more preferably less than about 20% chemicalprecursors or non-THAP-family or THAP-domain chemicals, still morepreferably less than about 10% chemical precursors or non-THAP-family orTHAP-domain chemicals, and most preferably less than about 5% chemicalprecursors or non-THAP-family or THAP-domain chemicals.

The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

Accordingly, another aspect of the invention pertains toanti-THAP-family or THAP-domain antibodies. The term “antibody” as usedherein refers to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site which specifically binds (immunoreacts with) anantigen, such as a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind a THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of a THAP-family orTHAP domain polypeptide. A monoclonal antibody composition thustypically displays a single binding affinity for a particularTHAP-family or THAP domain protein with which it immunoreacts.

PAR4

As mentioned above, Prostate apoptosis response-4 (PAR4) is a 38 kDaprotein initially identified as the product of a gene specificallyupregulated in prostate tumor cells undergoing apoptosis (for reviewssee Rangnekar, 1998; Mattson et al., 1999). The PAR4 nucleic acid andamino acid sequences, see Johnstone et al, Mol. Cell. Biol. 16 (12),6945-6956 (1996); and Genbank accession no. U63809 (SEQ ID NO: 118).

As used interchangeably herein, a “PAR4 activity”, “biological activityof a PAR4” or “functional activity of a PAR4”, refers to an activityexerted by a PAR4 protein, polypeptide or nucleic acid molecule asdetermined in vivo, or in vitro, according to standard techniques. Inone embodiment, a PAR4 activity is a direct activity, such as anassociation with a PAR4-target molecule or most preferably apoptosisinduction activity, or inhibition of cell proliferation or cell cycle.As used herein, a “target molecule” is a molecule with which a PAR4protein binds or interacts in nature, such that PAR4-mediated functionis achieved. An example of a PAR4 target molecule is a THAP-familyprotein such as THAP1 or THAP2, or a PML-NBs protein. A PAR4 targetmolecule can be a PAR4 protein or polypeptide or a non-PAR4 molecule.For example, a PAR4 target molecule can be a non-PAR4 protein molecule.Alternatively, a PAR4 activity is an indirect activity, such as anactivity mediated by interaction of the PAR4 protein with a PAR4 targetmolecule such that the target molecule modulates a downstream cellularactivity (e.g., interaction of a PAR4 molecule with a PAR4 targetmolecule can modulate the activity of that target molecule on anintracellular signaling pathway).

Binding or interaction with a PAR4 target molecule (such as THAP1/PAR4described herein) or with other targets can be detected for exampleusing a two hybrid-based assay in yeast to find drugs that disruptinteraction of the PAR4 bait with the target (e.g. PAR4) prey, or an invitro interaction assay with recombinant PAR4 and target proteins (e.g.THAP1 and PAR4).

Chemokines

Chemokines (chemoattractant cytokines) are small secreted polypeptidesof about 70-110 amino acids that regulate trafficking and effectorfunctions of leukocytes, and play an important role in inflammation andhost defence against pathogens (reviewed in Baggiolini M., et al. (1997)Annu. Rev. inmmunol. 15: 675-705; Proost P., et al. (1996) Int. J. Clin.Lab. Rse. 26: 211-223; Premack, et al. (1996) Nature Medicine 2:1174-1178; Yoshie, et al. (1997) J. Leukocyte Biol. 62: 634-644). Over45 different human chemokines have been described to date. They vary intheir specificities for different leukocyte types (neutrophils,monocytes, eosinophils, basophils, lymphocytes, dendritic cells, etc.),and in the types of cells and tissues where the chemokines aresynthesized. Chemokines are typically produced at sites of tissue injuryor stress, where they promote the infiltration of leukocytes intotissues and facilitate an inflammatory response. Some chemokines actselectively on immune system cells such as subsets of T-cells or Blymphocytes or antigen presenting cells, and may thereby promote immuneresponses to antigens. Some chemokines also have the ability to regulatethe growth or migration of hematopoietic progenitor and stem cells thatnormally differentiate into specific leukocyte types, thereby regulatingleukocyte numbers in the blood.

The activities of chemokines are mediated by cell surface receptorswhich are members of the family of seven transmembrane, G-proteincoupled receptors. At present, over fifteen different human chemokinereceptors are known, including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5. These receptorsvary in their specificites for specific chemokines. Some receptors bindto a single known chemokine, while others bind to multiple chemokines.Binding of a chemokine to its receptor typically induces intracellularsignaling responses such as a transient rise in cytosolic calciumconcentration, followed by cellular biological responses such aschemotaxis.

Chemokines are important in medicine because they regulate the movementand biological activities of leukocytes in many disease situations,including, but not limited to: allergic disorders, autoimmune diseases,ischemia/reperfusion injury, development of atherosclerotic plaques,cancer (including mobilization of hematopoietic stem cells for use inchemotherapy or myeloprotection during chemotherapy), chronicinflammatory disorders, chronic rejection of transplanted organs ortissue grafts, chronic myelogenous leukemia, and infection by HIV andother pathogens. Antagonists of chemokines or chemokine receptors may beof benefit in many of these diseases by reducing excessive inflammationand immune system responses.

The activity of chemokines is tightly regulated to prevent excessiveinflammation that can cause disease. Inhibition of chemokines byneutralizing antibodies in animal models (Sekido et al. (1993) Nature365:654-657) or disruption of mouse chemokine genes (Cook et al. (1995)Science 269:1583-1588) have confirmed a critical role of chemokines invivo in inflammation mediated by virus infection or other processes. Theproduction of soluble versions of cytokine receptors containing only theextracellular binding domain, represents a physiological and therapeuticstrategy to block the activity of some cytokines (Rose-John and Heinrich(1994) Biochem J. 300:281-290; Heaney and Golde (1996) Blood87:847-857). However, the seven transmembrane domain structure ofchemokine receptors makes the construction of soluble, inhibitoryreceptors difficult, and thus antagonists based on mutated chemokines,blocking peptides or antibodies are under evaluation as chemokineinhibitors (D'Souza & Harden (1996) Nature Medecine 2:1293-1300; Howardet al. (1996) Trends Biotech. 14:46-51; Baggiolini (1998) Nature392:565-568; Rollins (1997) Blood 90:909-928).

Several viral chemokine binding proteins have been described that may beuseful as soluble chemokine inhibitors. Soluble chemokine-bindingproteins have been previously detected in poxviruses. Firstly, themyxoma virus T7 protein, which was first identified as a soluble IFN-yReceptor (Upton et al. (1992) Science 258:1369-1372), binds to a rangeof chemokines through the heparin-binding domain and affects theinfiltration of cells into infected tissue (Lalani et al. (1997) J Virol71:4356-4363). The protein is described in U.S. Patent No. 5,834,419 andInternational Publication No. WO96/33730, and is designated CBP-1.Secondly, it was demonstrated that VV strain Lister expresses a soluble35 kDa protein that is secreted from infected cells and which binds manyCC chemokines (Graham et al. (1997) Virology 229:12-24; Smith et al.(1997) Virology 236:316-327; Alcami et al (1998) J Immunol 160:624-633),but not CXC chemokines, through a domain distinct from theheparin-binding domain (Smith et al. (1997) Virology 236:316-327; Alcamiet al (1998) J Immunol 160:624-633). This protein has been called vCKBP(Alcami et al (1998) J Immunol 160:624-633). The protein is alsodescribed in U.S. Patent No. 5,871,740 and International Publication No.WO97/11714. One main disadvantage to the use of these viral proteins ina clinical setting is that antigenicity severely limits theirindications. As such, there is a strong interest in the identificationof cellular chemokine-binding proteins.

Some aspects of the present invention relate to cellular polypeptidesand homologs thereof, portions of cellular polypeptides and homologsthereof as well as modified cellular polypeptides and homologs thereofthat bind to one or more chemokines. In some embodiments of the presentinvention such cellular polypeptides are THAP-family polypeptides,including THAP-1, chemokine-binding domains of THAP-family polypeptides(including a chemokine-binding domain of THAP-1), THAP-familypolypeptide or THAP-family chemokine-binding domain fusions toimmunoglobulin Fc (including THAP-1 fused to an immunoglobulin Fc regionor a chemokine-binding domain of THAP-1 fused to an immunoglobulin Fcregion), oligomers of THAP-family polypeptides or THAP-familychemokine-binding domains (including THAP-1 oligomers or oligomers of achemokine-binding domain of THAP-1), or homologs of any of theabove-listed compositions. Throughout this disclosure, the above-listedpolypeptides are referred to as THAP-type chemokine-binding agents. Eachof these THAP-type chemokine-binding agents are described in detailbelow.

SLC/CCL21 (SLC)

Biological Roles of SLC

The signals which mediate T-cell infiltration during T-cell auto-immunediseases are poorly understood. SLC/CCL21 (SEQ ID NO: 119) is highlypotent and highly specific for attracting T-cell migration. It wasinitially thought to be expressed only in secondary lymphoid organs,directing naive T-cells to areas of antigen presentation. However, usingimmunohistology it was found that expression of CCL21 was highly inducedin endothelial cells of T-cell auto-immune infiltrative skin diseases(Christopherson et al. (2002) Blood electronic publication prior toprinted publication). No other T-cell chemokine was consistently inducedin these T cell skin diseases. The receptor for CCL21, CCR7, was alsofound to be highly expressed on the infiltrating T-cells, the majorityof which expressed the memory CD45Ro phenotype. Inflamed venulesendothelial cells expressing SLC/CCL21 in T cell infiltrative autoimmuneskin diseases may therefore play a key role in the regulation of T-cellmigration into these tissues.

There are a number of other autoimmune diseases where induced expressionof SLC/CCL21 in endothelial cells may cause abnormal recruitment ofT-cells from the circulation to sites of pathologic inflammation. Forinstance, chemokine SLC/CCL21 appears to be important for aberrantT-cell infiltration in experimental autoimmune encephalomyelitis (EAE),an animal model for multiple sclerosis (Alt et al. (2002) Eur J Immunol32:2133-44). Migration of autoaggressive T cells across the blood-brainbarrier (BBB) is critically involved in the initiation of EAE. Thedirect involvement of chemokines in this process was suggested by theobservation that G-protein-mediated signaling is required to promoteadhesion strengthening of encephalitogenic T cells on BBB endothelium invivo. A search for chemokines present at the BBB, by in situhybridizations and immunohistochemistry revealed expression of thelymphoid chemokines CCL19/ELC and CCL21/SLC in venules surrounded byinflammatory cells (Alt et al. (2002) Eur J Immunol 32:2133-44). Theirexpression was paralleled by the presence of their common receptor CCR7in inflammatory cells in brain and spinal cord sections of miceafflicted with EAE. Encephalitogenic T cells showed surface expressionof CCR7 and specifically chemotaxed towards both CCL19 or CCL21 in aconcentration dependent and pertussis toxin-sensitive manner comparableto naive lymphocytes in vitro. Binding assays on frozen sections of EAEbrains demonstrated a functional involvement of CCL19 and CCL21 inadhesion strengthening of encephalitogenic T lymphocytes to inflamedvenules in the brain (Alt et al. (2002) Eur J Immunol 32:2133-44). Takentogether these data suggested that the lymphoid chemokines CCL19 andCCL21 besides regulating lymphocyte homing to secondary lymphoid tissueare involved in T lymphocyte migration into the immunoprivileged centralnervous system during immunosurveillance and chronic inflammation.

Other diseases where induced expression of SLC/CCL21 in venularendothelial cells has been observed include rheumatoid arthritis (Pageet al. (2002) J Immunol 168:5333-5341) and experimental autoimmunediabetes (Hjelmstrom et al. (2000) Am J Path 156:1133-1138). Therefore,chemokine SLC/CCL21 may be an important pharmacological target in T-cellauto-immune diseases. Inhibitors of SLC/CCL21 may be effective agents attreating these T cell infiltrative diseases by interfering with theabnormal recruitment of T cells, from the circulation to sites ofpathologic inflammation, by endothelial cells expressing SLC/CCL21. Thereduction in T cell migration into involved tissue would reduce theT-cell inflicted damage seen in those diseases.

Ectopic lymphoid tissue formation is a feature of many chronicinflammatory diseases, including rheumatoid arhtritis, inflammatorybowel diseases (Crohn's disease, ulcerative colitis), autoimmunediabetes, chronic inflammatory skin diseases (lichen panus, psoriasis, .. . ), Hashimoto's thyroiditis, Sjogren's syndrome, gastric lymphomasand chronic inflammatory liver disease (Girard and Springer (1995)Immunol today 16:449-457; Takemura et al. (2001) J Immunol167:1072-1080; Grant et al. (2002) Am J Pathol 2002 160:1445-55;Yoneyama et al. (2001) J Exp Med 193:35-49).

Ectopic expression of SLC/CCL21 has been shown to induce lymphoidneogenesis, both in mice and in human inflammatory diseases. In mice,transgenic expression of SLC/CCL21 in the pancreas (Fan et al. (2000) JImmunol 164:3955-3959; Chen et al. (2002) J Immunol 168:1001-1008;Luther et al. (2002) J Immunol 169:424433), a non-lymphoid tissue, hasbeen found to be sufficient for the development and organization ofectopic lymphoid tissue through differential recruitment of T and Blymphocytes and induction of high endothelial venules, specialized bloodvessels for lymphocyte migration (Girard and Springer (1995) Immunoltoday 16:449-457). In humans, hepatic expression of SLC/CCL21 has beenshown to promote the development of high endothelial venules andportal-associated lymphoid tissue in chronic inflammatory liver disease(Grant et al. (2002) Am J Pathol 2002 160:1445-55; Yoneyama et al.(2001) J Exp Med 193:35-49).The chronic inflammatory liver diseaseprimary sclerosing cholangitis (PSC) is associated with portalinflammation and the development of neolymphoid tissue in the liver.More than 70% of patients with PSC have a history of inflammatory boweldisease and strong induction of SLC/CCL21 on CD34(+) vascularendothelium in portal associated lymphoid tissue in PSC has beenreported (Grant et al. (2002) Am J Pathol 2002 160:1445-55). Incontrast, CCL21 is absent from LYVE-1(+) lymphatic vessel endothelium.Intrahepatic lymphocytes in PSC include a population of CCR7(+) T cellsonly half of which express CD45RA and which respond to CCL21 inmigration assays. The expression of CCL21 in association with mucosaladdressin cell adhesion molecule-1 in portal tracts in PSC may promotethe recruitment and retention of CCR7(+) mucosal lymphocytes leading tothe establishment of chronic portal inflammation and the expandedportal-associated lymphoid tissue. These findings are supported bystudies in an animal model of chronic hepatic inflammation, that haveshown that anti-SLC/CCL21 antibodies prevent the development of highendothelial venules and portal-associated lymphoid tissue (Yoneyama etal. (2001) J Exp Med 193:35-49).

Induction of chemokine SLC/CCL21 at a site of inflammation could convertthe lesion from an acute to a chronic state with correspondingdevelopment of ectopic lymphoid tissue. Blocking chemokine SLC/CCL21activity in chronic inflammatory diseases may therefore have significanttherapeutic value.

As used herein, “SLC/CCL21” and “SLC” are synonymous.

THAP-Family Members Comprising a THAP Domain

Based on the elucidation of a biological activity of the THAP1 proteinin apoptosis as described herein, the inventors have identified andfurther characterized a novel protein motif, referred to herein as THAPdomain. The THAP domain has been identified by the present inventors inseveral other polypeptides, as further described herein. Knowledge ofthe structure and function of the THAP domain allows the performing ofscreening assays that can be used in the preparation or screening ofmedicaments capable of modulating interaction with a THAP-family-targetmolecule, modulating cell cycle and cell proliferation, inducingapoptosis or enhancing or participating in the induction of apoptosis.

As used interchangeably herein, a THAP-family protein or polypeptide, ora THAP-family member refers to any polypeptide having a THAP domain asdescribed herein. As mentioned, the inventors have provided severalspecific THAP-family members. Thus, as referred to herein, a THAP-familyprotein or polypeptide, or a THAP-family member, includes but is notlimited to a THAP-0, THAP1, THAP-2, THAP-3, THAP-4, THAP-5, TRAP-6,THAP-7, TRAP-8, THAP-9, THAP10 or a THAP11 polypeptide.

As used interchangeably herein, a “THAP-family activity”, “biologicalactivity of a THAP-family member” or “functional activity of aTHAP-family member”, refers to an activity exerted by a THAP family orTHAP domain polypeptide or nucleic acid molecule, or a biologicallyactive fragment or homologue thereof comprising a THAP as determined invivo, or in vitro, according to standard techniques. In one embodiment,a THAP-family activity is a direct activity, such as an association witha THAP-family-target molecule or most preferably apoptosis inductionactivity, or inhibition of cell proliferation or cell cycle. As usedherein, a “THAP-family target molecule” is a molecule with which aTHAP-family protein binds or interacts in nature, such that a THAPfamily-mediated function is achieved. For example, a THAP family targetmolecule can be another THAP-family protein or polypeptide which issubstantially identical or which shares structural similarity (e.g.forming a dimer or multimer). In another example, a THAP family targetmolecule can be a non-THAP family comprising protein molecule, or anon-self molecule such as for example a Death Domain receptor. Bindingor interaction with a THAP family target molecule (such as THAP1/PAR4described herein) or with other targets can be detected for exampleusing a two hybrid-based assay in yeast to find drugs that disruptinteraction of the THAP family bait with the target (e.g. PAR4) prey, oran in vitro interaction assay with recombinant THAP family and targetproteins (e.g. THAP1 and PAR4). In yet another example, a THAP familytarget molecule can be a nucleic acid molecule. For instance, a THAPfamily target molecule can be DNA.

Alternatively, a THAP-family activity may be an indirect activity, suchas an activity mediated by interaction of the TRAP-family protein with aTHAP-family target molecule such that the target molecule modulates adownstream cellular activity (e.g., interaction of a THAP-familymolecule with a THAP-family target molecule can modulate the activity ofthat target molecule on an intracellular signaling pathway).

THAP-family activity is not limited to the induction of apoptoticactivity, but may also involve enhancing apoptotic activity. As deathdomains may mediate protein-protein interactions, including interactionswith other death domains, THAP-family activity may involve transducing acytocidal signal.

Assays to detect apoptosis are well known. In a preferred example, anassay is based on serum-withdrawal induced apoptosis in a 3T3 cell linewith tetracycline-regulated expression of a THAP family membercomprising a THAP domain. Other non-limiting examples are alsodescribed.

In one example, a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof can be the minimumregion of a polypeptide that is necessary and sufficient for thegeneration of cytotoxic death signals. Exemplary assays for apoptosisactivity are further provided herein.

In specific embodiments, PAR4 is a preferred THAP1 and/or THAP2 targetmolecule. In another aspect, a THAP1 target molecule is a PML-NBprotein.

In further aspects, THAP-domain or a THAP-family polypeptide comprises aDNA binding domain.

In other aspects, a THAP-family activity is detected by assessing any ofthe following activities: (1) mediating apoptosis or cell proliferationwhen expressed in or introduced into a cell, most preferably inducing orenhancing apoptosis, and/or most preferably reducing cell proliferation;(2) mediating apoptosis or cell proliferation of an endothelial cell;(3) mediating apoptosis or cell proliferation of a hyperproliferativecell; (4) mediating apoptosis or cell proliferation of a CNS cell,preferably a neuronal or glial cell; (5) an activity determined in ananimal selected from the group consisting of mediating, preferablyinhibiting angiogenesis, mediating, preferably inhibiting inflammation,inhibition of metastatic potential of cancerous tissue, reduction oftumor burden, increase in sensitivity to chemotherapy or radiotherapy,killing a cancer cell, inhibition of the growth of a cancer cell, orinduction of tumor regression; or (6) interaction with a THAP familytarget molecule or THAP domain target molecule, preferably interactionwith a protein or a nucleic acid. Detecting THAP-family activity mayalso comprise detecting any suitable therapeutic endpoint discussedherein in the section titled “Methods of Treatment”. THAP-familyactivity may be assessed either in vitro (cell or non-cell based) or invivo depending on the assay type and format.

A THAP domain has been identified in the N-terminal region of the THAP1protein, from about amino acid 1 to about amino acid 89 of SEQ ID NO: 3based on sequence analysis and functional assays. A THAP domain has alsobeen identified in THAP2 to THAP0 of SEQ ID NOs: 4-14. However, it willbe appreciated that a functional THAP domain may be only a small portionof the protein, about 10 amino acids to about 15 amino acids, or fromabout 20 amino acids to about 25 amino acids, or from about 30 aminoacids to about 35 amino acids, or from about 40 amino acids to about 45amino acids, or from about 50 amino acids to about 55 amino acids, orfrom about 60 amino acids to about 70 amino acids, or from about 80amino acids to about 90 amino acids, or about 100 amino acids in length.Alternatively, THAP domain or THAP family polypeptide activity, asdefined above, may require a larger portion of the native protein thanmay be defined by protein-protein interaction, DNA binding, cell assaysor by sequence alignment. A portion of a THAP domain-containingpolypeptide from about 10 amino acids to about 115 amino acids, or fromabout 120 amino acids to 130 amino acids, or from about 140 amino acidsto about 150 amino acids, or from about 160 amino acids to about 170amino acids, or from about 180 amino acids to about 190 amino acids, orfrom about 200 amino acids to about 250 amino acids, or from about 300amino acids to about 350 amino acids, or from about 400 amino acids toabout 450 amino acids, or from about 500 amino acids to about 600 aminoacids, to the extent that said length is consistent with the SEQ ID No,or the full length protein, for example any full length protein in SEQID NOs: 1-114, may be required for function.

As discussed, the invention includes a novel protein domain, includingseveral examples of THAP-family members. The invention thus encompassesa THAP-family member comprising a polypeptide having at least a THAPdomain sequence in the protein or corresponding nucleic acid molecule,preferably a THAP domain sequence corresponding to SEQ ID NOs: 1-2. ATHAP-family member may comprise an amino acid sequence of at least about25, 30, 35, 40, 45, 50, 60, 70, 80 to 90 amino acid residues in length,of which at least about 50-80%, preferably at least about 60-70%, morepreferably at least about 65%, 75% or 90% of the amino acid residues areidentical or similar amino acids-to the THAP consensus domain SEQ IDNOs: 1-2.

Identity or similarity may be determined using any desired algorithm,including the algorithms and parameters for determining homology whichare described herein.

Optionally, a THAP-domain-containing THAP-family polypeptide comprises anuclear localization sequence (NLS). As used herein, the term nuclearlocalization sequence refers to an amino sequence allowing theTHAP-family polypeptide to be localized or transported to the cellnucleus. A nuclear localization sequence generally comprises at leastabout 10, preferably about 13, preferably about 16, more preferablyabout 19, and even more preferably about 21, 23, 25, 30, 35 or 40 aminoacid residues. Alternatively, a THAP-family polypeptide may comprise adeletion of part or the entire NLS or a substitution or insertion in aNLS sequence, such that the modified THAP-family polypeptide is notlocalized or transported to the cell nucleus.

Isolated proteins of the present invention, preferably THAP family orTHAP domain polypeptides, or a biologically active fragments orhomologues thereof, have an amino acid sequence sufficiently homologousto the consensus amino acid sequence of SEQ ID NOs: 1-2. As used herein,the term “sufficiently homologous” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains have at least about30-40% identity, preferably at least about 40-50% identity, morepreferably at least about 50-60%, and even more preferably at leastabout 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identityacross the amino acid sequences of the domains and contain at least oneand preferably two structural domains or motifs, are defined herein assufficiently homologous. Furthermore, amino acid or nucleotide sequenceswhich share at least about 30%, preferably at least about 40%, morepreferably at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or99.8% identity and share a common functional activity are defined hereinas sufficiently homologous.

It be appreciated that the invention encompasses any of the THAP-familypolypeptides, as well as fragment thereof, nucleic acids complementarythereto and nucleic acids capable of hybridizing thereto under stringentconditions.

THAP-0 to THAP11

As mentioned, the inventors have identified several THAP-family members,including THAP-0, THAP1, THAP-2, THAP-3, THAP-4, THAP-5, THAP-6, THAP-7,THAP-8, THAP-9, THAP10 and THAP11.

THAP1 Nucleic Acids

The human THAP1 coding sequence, which is approximately 639 nucleotidesin length shown in SEQ ID NO: 160, encodes a protein which isapproximately 213 amino acid residues in length. One aspect of theinvention pertains to purified or isolated nucleic acid molecules thatencode THAP1 proteins or biologically active portions thereof as furtherdescribed herein, as well as nucleic acid fragments thereof. Saidnucleic acids may be used for example in therapeutic methods and drugscreening assays as further described herein.

The human THAP1 gene is localized at chromosomes 8, 18, 11.

The THAP1 protein comprises a THAP domain at amino acids 1-89, the roleof which in apoptosis is further demonstrated herein. The THAP1 proteincomprises an interferon gamma homology motif at amino acids 136-169 ofhuman THAP1 (NYTVEDTMHQRKRIHQLEQQVEKLRKKLKTAQQR) (SEQ ID NO: 178),exhibiting 41% identity in a 34 residue overlap with human interferongamma (amino acids 98-131). PML-NBs are closely linked to IFNgamma, andmany PML-NB components are induced by IFNgamma, with IFN gammaresponsive elements in the promoters of the corresponding genes. TheTHAP1 protein also includes a nuclear localization sequence at aminoacids 146-165 of human THAP1 (RKRIHQLEQQVEKLRKKLKT) (SEQ ID NO: 179).This sequence is responsible for localization of THAP1 in the nucleus.As demonstrated in the examples provided herein, deletion mutants ofTHAP1 lacking this sequence are no longer localized in the cell nucleus.The THAP1 protein further comprises a PAR4 binding motif (LE(X)₁₄QRXRRQXR(X)iiQRIKE) (SEQ ID NO: 180). The core of this motif has beendefined experimentally by site directed mutagenesis and by comparisonwith mouse ZIP/DAP-like kinase (another PAR4 binding partner) itoverlaps amino acids 168-175 of human THAP1 but the motif may alsoinclude a few residues upstream and downstream.

ESTs corresponding to THAP1 have been identified, and may bespecifically included or excluded from the nucleic acids of theinvention. The ESTs, as indicated below by accession number, provideevidence for tissue distribution for THAP1 as follows: AL582975 (B cellsfrom Burkitt lymphoma); BG708372 (Hypothalamus); BG563619 (liver);BG497522 (adenocarcinoma); BG616699 (liver); BE932253 (head_neck);AL530396 (neuroblastoma cells).

An object of the invention is a purified, isolated, or recombinantnucleic acid comprising the nucleotide sequence of SEQ ID NO: 160,complementary sequences thereto, and fragments thereof. The inventionalso pertains to a purified or isolated nucleic acid comprising apolynucleotide having at least 95% nucleotide identity with apolynucleotide of SEQ ID NO: 160, advantageously 99 % nucleotideidentity, preferably 99.5% nucleotide identity and most preferably 99.8%nucleotide identity with a polynucleotide of SEQ ID NO: 160, or asequence complementary thereto or a biologically active fragmentthereof. Another object of the invention relates to purified, isolatedor recombinant nucleic acids comprising a polynucleotide thathybridizes, under the stringent hybridization conditions defined herein,with a polynucleotide of SEQ ID NO: 160, or a sequence complementarythereto or a variant thereof or a biologically active fragment thereof.In further embodiments, nucleic acids of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID NO: 160, or the complementsthereof.

Also encompassed is a purified, isolated, or recombinant nucleic acidpolynucleotide encoding a THAP1 polypeptide of the invention, as furtherdescribed herein.

In another preferred aspect, the invention pertains to purified orisolated nucleic acid molecules that encode a portion or variant of aTHAP1 protein, wherein the portion or variant displays a THAP1 activityof the invention. Preferably said portion or variant is a portion orvariant of a naturally occurring full-length THAP1 protein. In oneexample, the invention provides a polynucleotide comprising, consistingessentially of, or consisting of a contiguous span of at least 12, 15,18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides of SEQ ID NO: 160, wherein said nucleic acid encodes a THAP1portion or variant having a THAP1 activity described herein. In otherembodiments, the invention relates to a polynucleotide encoding a THAP1portion consisting of 8-20, 20-50, 50-70, 60-100, 100 -150, 150-200,200-205 or 205-212 amino acids of SEQ ID NO: 3, or a variant thereof,wherein said THAP1 portion displays a THAP1 activity described herein.

The sequence of SEQ ID NO: 160 corresponds to the human THAP1 cDNA. ThiscDNA comprises sequences encoding the human THAP1 protein (i.e., “thecoding region”, from nucleotides 202 to 840, as well as 5′ untranslatedsequences (nucleotides 1-201) and 3′ untranslated sequences (nucleotides841 to 2173).

Also encompassed by the THAP1 nucleic acids of the invention are nucleicacid molecules which are complementary to THAP1 nucleic acids describedherein. Preferably, a complementary nucleic acid is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO: 160, suchthat it can hybridize to the nucleotide sequence shown in SEQ ID NO:160, thereby forming a stable duplex.

Another object of the invention is a purified, isolated, or recombinantnucleic acid encoding a THAP1 polypeptide comprising, consistingessentially of, or consisting of the amino acid sequence of SEQ ID NO:3, or fragments thereof, wherein the isolated nucleic acid moleculeencodes one or more motifs selected from the group consisting of a THAPdomain, a THAP1 target binding region, a nuclear localization signal anda interferon gamma homology motif. Preferably said THAP1 target bindingregion is a PAR4 binding region or a DNA binding region. For example,the purified, isolated or recombinant nucleic acid may comprise agenomic DNA or fragment thereof which encodes the polypeptide of SEQ IDNO: 3 or a fragment thereof or a cDNA consisting of, consistingessentially of, or comprising the sequence of SEQ ID NO: 160 orfragments thereof, wherein the isolated nucleic acid molecule encodesone or more motifs selected from the group consisting of a THAP domain,a THAP1 -target binding region, a nuclear localization signal and ainterferon gamma homology motif. Any combination of said motifs may alsobe specified. Preferably said THAP1 target binding region is a PAR4binding region or a DNA binding region. Particularly preferred nucleicacids of the invention include isolated, purified, or recombinant THAP1nucleic acids comprising, consisting essentially of, or consisting of acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200 or 300 nucleotides of a sequence selected from thegroup consisting of nucleotide positions ranges consisting of 607 to708, 637 to 696 and 703 to 747 of SEQ ID NO: 160. In preferredembodiments, a THAP1 nucleic acid encodes a THAP1 polypeptide comprisingat least two THAP1 functional domains, such as for example a THAP domainand a PAR4 binding region.

In further preferred embodiments, a THAP1 nucleic acid comprises anucleotide sequence encoding a THAP domain having the consensus aminoacid sequence of the formula of SEQ ID NOs: 1-2. A THAP1 nucleic acidmay also encode a THAP domain wherein at least about 95%, 90%, 85%,50-80%, preferably at least about 60-70%, more preferably at least about65% of the amino acid residues are identical or similar amino acids-tothe THAP domain consensus sequence (SEQ ID NOs: 1-2). The presentinvention also embodies isolated, purified, and recombinantpolynucleotides which encode a polypeptide comprising a contiguous spanof at least 6 amino acids, preferably at least 8 or 10 amino acids, morepreferably at least 15, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90 aminoacids according to the formula of SEQ ID NO: 1-2.

The nucleotide sequence determined from the cloning of the THAP1geneallows for the generation of probes and primers designed for use inidentifying and/or cloning other THAP1 family members (e.g. sharing thenovel functional domains), as well as THAP1 homologues from otherspecies.

A nucleic acid fragment encoding a “biologically active portion of aTHAP1 protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO: 160, which encodes a polypeptide having a THAP1biological activity (the biological activities of the THAP1 proteinsdescribed herein), expressing the encoded portion of the THAP1 protein(e.g., by recombinant expression in vitro or in vivo) and assessing theactivity of the encoded portion of the THAP1 protein.

The invention further encompasses nucleic acid molecules that differfrom the THAP1 nucleotide sequences of the invention due to degeneracyof the genetic code and encode the same THAP1 proteins and fragment ofthe invention.

In addition to the THAP1 nucleotide sequences described above, it willbe appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theTHAP1 proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism may exist among individualswithin a population due to natural allelic variation. Such naturalallelic variations can typically result in 1-5% variance in thenucleotide sequence of a THAP1 gene.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the THAP1 nucleic acids of the invention can be isolatedbased on their homology to the THAP1 nucleic acids disclosed hereinusing the cDNAs disclosed herein, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions.

Probes based on the THAP1 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a THAP1 protein, such as by measuring a level ofa THAP1-encoding nucleic acid in a sample of cells from a subject e.g.,detecting THAP1 mRNA levels or determining whether a genomic THAP1 genehas been mutated or deleted.

THAP1 Polypeptides

The term “THAP1 polypeptides” is used herein to embrace all of theproteins and polypeptides of the present invention. Also forming part ofthe invention are polypeptides encoded by the polynucleotides of theinvention, as well as fusion polypeptides comprising such polypeptides.The invention embodies THAP1 proteins from humans, including isolated orpurified THAP1 proteins consisting of, consisting essentially of, orcomprising the sequence of SEQ ID NO: 3.

The invention concerns the polypeptide encoded by a nucleotide sequenceof SEQ ID NO: 160, a complementary sequence thereof or a fragmentthereto.

The present invention embodies isolated, purified, and recombinantpolypeptides comprising a contiguous span of at least 6 amino acids,preferably at least 8 to 10 amino acids, more preferably at least 12,15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID NO: 3. In otherpreferred embodiments the contiguous stretch of amino acids comprisesthe site of a mutation or functional mutation, including a deletion,addition, swap or truncation of the amino acids in the THAP1 proteinsequence. The invention also concerns the polypeptide encoded by theTHAP1 nucleotide sequences of the invention, or a complementary sequencethereof or a fragment thereof.

One aspect of the invention pertains to isolated THAP1 proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-THAP1 antibodies. In oneembodiment, native THAP1 proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, THAP1 proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a THAP1 protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

Typically, biologically active portions comprise a domain or motif withat least one activity of the THAP1 protein. The present invention alsoembodies isolated, purified, and recombinant portions or fragments ofone THAP1 polypeptide comprising a contiguous span of at least 6 aminoacids, preferably at least 8 to 10 amino acids, more preferably at least12, 15, 20, 25, 30, 40, 50, 100 or 200 amino acids of SEQ ID NO: 3. Alsoencompassed are THAP1 polypeptide which comprise between 10 and 20,between 20 and 50, between 30 and 60, between 50 and 100, or between 100and 200 amino acids of SEQ ID NO: 3. In other preferred embodiments thecontiguous stretch of amino acids comprises the site of a mutation orfunctional mutation, including a deletion, addition, swap or truncationof the amino acids in the THAP1 protein sequence.

A biologically active THAP1 protein may, for example, comprise at least1, 2, 3, 5, 10, 20 or 30 amino acid changes from the sequence of SEQ IDNO: 3, or may encode a biologically active THAP1 protein comprising atleast 1%, 2%, 3%, 5%, 8%, 10% or 15% changes in amino acids from thesequence of SEQ ID NO: 3.

In a preferred embodiment, the THAP1 protein comprises, consistsessentially of, or consists of a THAP domain at amino acid positions 1to 89 shown in SEQ ID NO: 3, or fragments or variants thereof. In otheraspects, a THAP1 polypeptide comprises a THAP1-target binding region, anuclear localization signal and/or a Interferon Gamma Homology Motif.Preferably a THAP1 target binding region is a PAR4 binding region or aDNA binding region. The invention also concerns the polypeptide encodedby the THAP1 nucleotide sequences of the invention, or a complementarysequence thereof or a fragment thereof. The present invention thus alsoembodies isolated, purified, and recombinant polypeptides comprising,consisting essentially of or consisting of a contiguous span of at least6 amino acids, preferably at least 8 to 10 amino acids, more preferablyat least 12, 15, 20, 25, 30, 40, 50, 70, 80, 90 or 100 amino acids of anamino acid sequence selected from the group consisting of positions 1 to90, 136 to 169, 146 to 165 and 168 to 175 of SEQ ID NO: 3. In anotheraspect, a THAP1 polypeptide may encode a THAP domain wherein at leastabout 95%, 90%, 85%, 50-80%, preferably at least about 60-70%, morepreferably at least about 65% of the amino acid residues are identicalor similar amino acids—to the THAP domain consensus sequence (SEQ IDNOs: 1-2). Also encompassed by the present invention are isolated,purified, nucleic acids encoding a THAPl polypeptide comprising,consisting essentially of, or consisting of a THAP domain at amino acidpositions 1 to 90 shown in SEQ ID NO: 3, or fragments or variantsthereof.

In other embodiments, the THAP1 protein is substantially homologous tothe sequences of SEQ ID NO: 3, and retains the functional activity ofthe THAP1 protein, yet differs in amino acid sequence due to naturalallelic variation or mutagenesis, as described further herein.Accordingly, in another embodiment, the THAP1 protein is a protein whichcomprises an amino acid sequence shares more than about 60% but lessthan 100% homology with the amino acid sequence of SEQ ID NO: 3 andretains the functional activity of the THAP1 proteins of SEQ ID NO: 3,respectively. Preferably, the protein is at least about 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 99.8% homologous toSEQ ID NO: 3, but is not identical to SEQ ID NO: 3. Preferably the THAP1is less than identical (e.g. 100% identity) to a naturally occurringTHAP1. Percent homology can be determined as further detailed above.

THAP-2 to THAP11 and THAP-0 Nucleic Acids

As mentioned, the invention provides several members of the THAP-family.THAP-2, THAP-3, THAP-4, THAP-5, THAP-6, THAP-7, THAP-8, THAP-9, THAP10,THAP11 and THAP-0 are described herein. The human and mouse nucleotidesequences corresponding to the human cDNA sequences are listed in SEQ IDNOs: 161-171; and the human amino acid sequences are listed respectivelyin SEQ ID NOs: 4-14. Also encompassed by the invention are orthologs ofsaid THAP-family sequences, including mouse, rat, pig and otherorthologs, the amino acid sequences of which are listed in SEQ ID NOs:16-114 and the cDNA sequences are listed in SEQ ID NOs: 172-175.

THAP-2

The human THAP-2 cDNA, which is approximately 1302 nucleotides in lengthshown in SEQ ID NO: 161, encodes a protein which is approximately 228amino acid residues in length, shown in SEQ ID NO: 4. One aspect of theinvention pertains to purified or isolated nucleic acid molecules thatencode THAP-2 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP-2 geneis localized at chromosomes 12 and 3. The THAP-2 protein comprises aTHAP domain at amino acids 1 to 89. Analysis of expressed sequences(accession numbers indicated, which may be specifically included orexcluded from the nucleic acids of the invention) in databases suggeststhat THAP-2 is expressed as follows: BG677995 (squamous cell carcinoma);AV718199 (hypothalamus); BI600215 (hypothalamus); AI208780(Soares_testis_NHT); BE566995 (carcinoma cell line); AI660418 (thymuspooled)

THAP-3

The human THAP-3 cDNA which is approximately 1995 nucleotides in lengthshown in SEQ ID NO: 162. The THAP-3 gene encodes a protein which isapproximately 239 amino acid residues in length, shown in SEQ ID NO: 5.One aspect of the invention pertains to purified or isolated nucleicacid molecules that encode THAP-3 proteins or biologically activeportions thereof as further described herein, as well as nucleic acidfragments thereof. Said nucleic acids may be used for example intherapeutic methods and drug screening assays as further describedherein. The human THAP-3 gene is localized at chromosome 1. The THAP-3protein comprises a THAP domain at amino acids 1 to 89. Analysis ofexpressed sequences (accession numbers indicated, which may bespecifically included or excluded from the nucleic acids of theinvention) in databases suggests that THAP-3 is expressed as follows:BG700517 (hippocampus); BI460812 (testis); BG707197 (hypothalamus);AW960428 (−); BG437177 (large cell carcinoma); BE962820(adenocarcinoma); BE548411 (cervical carcinoma cell line); AL522189(neuroblastoma cells); BE545497 (cervical carcinoma cell line); BE280538(choriocarcinoma); BI086954 (cervix); BE744363 (adenocarcinoma cellline); and BI549151 (hippocampus).

THAP-4

The human THAP-4cDNA, shown as a sequence having 1999 nucleotides inlength shown in SEQ ID NO: 163, encodes a protein which is approximately577 amino acid residues in length, shown in SEQ ID NO: 6. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-4 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The THAP-4 proteincomprises a THAP domain at amino acids 1 to 90. Analysis of expressedsequences (accession numbers indicated, which may be specificallyincluded or excluded from the nucleic acids of the invention) indatabases suggests that THAP-4 is expressed as follows: AL544881(placenta); BE384014 (melanotic melanoma); AL517205 (neuroblastomacells); BG394703 (retinoblastoma); BG472327 (retinoblastoma); BI196071(neuroblastoma); BE255202 (retinoblastoma); BI017349 (lung_jumor);BF972153 (leiomyosarcoma cell line); BG116061 (duodenal adenocarcinomacell line); AL530558 (neuroblastoma cells); AL520036 (neuroblastomacells); AL559902 (B cells from Burkitt lymphoma); AL534539 (Fetalbrain); BF686560 (leiomyosarcoma cell line); BF345413 (anaplasticoligodendroglioma with 1p/19q loss); BG117228 (adenocarcinoma cellline); BG490646 (large cell carcinoma); and BF769104 (epid_tumor).

THAP-5

The human THAP-5 cDNA, shown as a sequence having 1034 nucleotides inlength shown in SEQ ID NO: 164, encodes a protein which is approximately239 amino acid residues in length, shown in SEQ ID NO: 7. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-5 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP-5 geneis localized at chromosome 7. The THAP-5 protein comprises a THAP domainat amino acids 1 to 90. Analysis of expressed sequences (accessionnumbers indicated, which may be specifically included or excluded fromthe nucleic acids of the invention) in databases suggests that THAP-5 isexpressed as follows: BG575430 (mammary adenocarcinoma cell line);BI545812 (hippocampus); BI560073 (testis); BG530461 (embryonalcarcinoma); BF244164 (glioblastoma); BI461364 (testis); AW407519(germinal center B cells); BF103690 (embryonal carcinoma); and BF939577(kidney).

THAP-6

The human THAP-6cDNA, shown as a sequence having 2291 nucleotides inlength shown in SEQ ID NO: 165, encodes a protein which is approximately222 amino acid residues in length, shown in SEQ ID NO: 8. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-6 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP-6 geneis localized at chromosome 4. The THAP-6 protein comprises a THAP domainat amino acids 1 to 90. Analysis of expressed sequences (accessionnumbers indicated, which may be specifically included or excluded fromthe nucleic acids of the invention) in databases suggests that THAP-6 isexpressed as follows: AV684783 (hepatocellular carcinoma); AV698391(hepatocellular carcinoma); BI560555 (testis); AV688768 (hepatocellularcarcinoma); AV692405 (hepatocellular carcinoma); and AV696360(hepatocellular carcinoma).

THAP-7

The human THAP-7 cDNA, shown as a sequence having 1242 nucleotides inlength shown in SEQ ID NO: 166, encodes a protein which is approximately309 amino acid residues in length, shown in SEQ ID NO: 9. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-7 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP-7 geneis localized at chromosome 22q11.2. The THAP-7 protein comprises a THAPdomain at amino acids 1 to 90. Analysis of expressed sequences(accession numbers indicated, which may be specifically included orexcluded from the nucleic acids of the invention) in databases suggeststhat THAP-7 is expressed as follows: BI193682 (epithelioid carcinomacell line); BE253146 (retinoblastoma); BE622113 (melanotic melanoma);BE740360 (adenocarcinoma cell line); BE513955 (Burkitt lymphoma);AL049117 (testis); BF952983 (nervous_normal); AW975614 (−); BE273270(renal cell adenocarcinoma); BE738428 (glioblastoma); BE388215(endometrium adenocarcinoma cell line); BF762401 (colon_est); andBG329264 (retinoblastoma).

THAP-8

The human THAP-8 cDNA, shown as a sequence having 1383 nucleotides inlength shown in SEQ ID NO: 167, encodes a protein which is approximately274 amino acid residues in length, shown in SEQ ID NO: 10. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-8 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP-8 geneis localized at chromosome 19. The THAP-8 protein comprises a THAPdomain at amino acids 1 to 92. Analysis of expressed sequences(accession numbers indicated, which may be specifically included orexcluded from the nucleic acids of the invention) in databases suggeststhat THAP-8 is expressed as follows: BG703645 (hippocampus); BF026346(melanotic melanoma); BE728495 (melanotic melanoma); BG334298 (melanoticmelanoma); and BE390697 (endometrium adenocarcinoma cell line).

THAP-9

The human THAP-9 cDNA, shown as a sequence having 693 nucleotides inlength shown in SEQ ID NO: 168, encodes a protein which is approximately231 amino acid residues in length, shown in SEQ ID NO: 11. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-9 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The THAP-9 proteincomprises a THAP domain at amino acids 1 to 92. Analysis of expressedsequences (accession numbers indicated, which may be specificallyincluded or excluded from the nucleic acids of the invention) indatabases suggests that THAP-9 is expressed as follows: AA333595 (Embryo8 weeks).

THAP10

The human THAP10 cDNA, shown as a sequence having 771 nucleotides inlength shown in SEQ ID NO: 169, encodes a protein which is approximately257 amino acid residues in length, shown in SEQ ID NO: 12. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP10 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP10 geneis localized at chromosome 15. The THAP10 protein comprises a THAPdomain at amino acids 1 to 90. Analysis of expressed sequences(accession numbers indicated, which may be specifically included orexcluded from the nucleic acids of the invention) in databases suggeststhat THAP10 is expressed as follows: AL526710 (neuroblastoma cells);AV725499 (Hypothalamus); AW966404 (−); AW296810 (lung); and AL557817 (Tcells from T cell leukemia).

THAP11

The human THAP11 cDNA, shown as a sequence having 942 nucleotides inlength shown in SEQ ID NO: 170, encodes a protein which is approximately314 amino acid residues in length, shown in SEQ ID NO: 13. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP11proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP11geneis localized at chromosome 16. The THAP11 protein comprises a THAPdomain at amino acids 1 to 90. Analysis of expressed sequences(accession numbers indicated, which may be specifically included orexcluded from the nucleic acids of the invention) in databases suggeststhat THAP11is expressed as follows: AU142300 (retinoblastoma); B1261822(lymphoma cell line); BG423102 (renal cell adenocarcinoma); and BG423864(kidney).

THAP-0

The human THAP-0 cDNA, shown as a sequence having 2283 nucleotides inlength shown in SEQ ID NO: 171, encodes a protein which is approximately761 amino acid residues in length, shown in SEQ ID NO: 14. One aspect ofthe invention pertains to purified or isolated nucleic acid moleculesthat encode THAP-0 proteins or biologically active portions thereof asfurther described herein, as well as nucleic acid fragments thereof.Said nucleic acids may be used for example in therapeutic methods anddrug screening assays as further described herein. The human THAP-0 geneis localized at chromosome 11. The THAP-0 protein comprises a THAPdomain at amino acids 1 to 90. Analysis of expressed sequences(accession numbers indicated, which may be specifically included orexcluded from the nucleic acids of the invention) in databases suggeststhat THAP-0 is expressed as follows: BE713222 (head neck); BE161184(head_neck); AL119452 (amygdala); AU129709 (teratocarcinoma); AW965460(−); AW965460(−); AW958065 (−); and BE886885 (leiomyosarcoma).

An object of the invention is a purified, isolated, or recombinantnucleic acid comprising the nucleotide sequence of SEQ ID NOs: 161-171,173-175 or complementary. sequences thereto, and fragments thereof. Theinvention also pertains to a purified or isolated nucleic acidcomprising a polynucleotide having at least 95% nucleotide identity witha polynucleotide of SEQ ID NOs: 161-171 or 173-175, advantageously 99 %nucleotide identity, preferably 99.5% nucleotide identity and mostpreferably 99.8% nucleotide identity with a polynucleotide of SEQ IDNOs: 161-171, 173-175 or a sequence complementary thereto or abiologically active fragment thereof. Another object of the inventionrelates to purified, isolated or recombinant nucleic acids comprising apolynucleotide that hybridizes, under the stringent hybridizationconditions defined herein, with a polynucleotide of SEQ ID NOs: 161-171,173-175 or a sequence complementary thereto or a variant thereof or abiologically active fragment thereof. In further embodiments, nucleicacids of the invention include isolated, purified, or recombinantpolynucleotides comprising a contiguous span of at least 12, 15, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides of a sequence selected from the group consisting of SEQ IDNOs: 161-171, 173-175 or the complements thereof.

Also encompassed is a purified, isolated, or recombinant nucleic acidpolynucleotide encoding a THAP-2 to THAP11 or THAP-0 polypeptide of theinvention, as further described herein.

In another preferred aspect, the invention pertains to purified orisolated nucleic acid molecules that encode a portion or variant of aTHAP-2 to THAP11 or THAP-0 protein, wherein the portion or variantdisplays a THAP-2 to THAP11 or THAP-0 activity of the invention.Preferably said portion or variant is a portion or variant of anaturally occurring full-length THAP-2 to THAP11 or THAP-0 protein. Inone example, the invention provides a polynucleotide comprising,consisting essentially of, or consisting of a contiguous span of atleast 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides, to the extent that the length of said span isconsistent with the length of the SEQ ID NO, of a sequence selected fromthe group consisting of SEQ ID NOs: 161-171, 173-175, wherein saidnucleic acid encodes a THAP-2 to THAP11 or THAP-0 portion or varianthaving a THAP-2 to THAP11 or THAP-0 activity described herein. In otherembodiment, the invention relates to a polynucleotide encoding a THAP-2to THAP11 or THAP-0 portion consisting of 8-20, 20-50, 50-70, 60-100,100-150, 150-200, 200-250 or 250-350 amino acids, to the extent that thelength of said portion is consistent with the length of the SEQ ID NO:of a sequence selected from the group consisting of SEQ ID NOs: 4-14,17-21, 23-40, 42-56, 58-98, 100-114 or a variant thereof, wherein saidTHAP-2 to THAP11 or THAP-0 portion displays a THAP-2 to THAP11 or THAP-0activity described herein.

A THAP-2 to THAP11 or THAP-0 variant nucleic acid may, for example,encode a biologically active THAP-2 to THAP11 or THAP-0 proteincomprising at least 1, 2, 3, 5, 10, 20 or 30 amino acid changes from therespective sequence selected from the group consisting of SEQ ID NO:4-14, 17-21, 23-40, 42-56, 58-98 and 100-114 or may encode abiologically active THAP-2 to THAP11 or THAP-0 protein comprising atleast 1%, 2%, 3%, 5%, 8%, 10% or 15% changes in amino acids from therespective sequence of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 and100-114.

The sequences of SEQ ID NOs: 4-14 correspond to the human THAP-2 toTHAP11 and THAP-0 DNAs respectively. SEQ ID NOs: 17-21, 23-40, 42-56,58-98, 100-114 correspond to mouse, rat, pig and other orthologs.

Also encompassed by the THAP-2 to THAP11 and THAP-0 nucleic acids of theinvention are nucleic acid molecules which are complementary to THAP-2to THAP11 or THAP-0 nucleic acids described herein. Preferably, acomplementary nucleic acid is sufficiently complementary to thenucleotide respective sequence shown in SEQ ID NOs: 161-171 and 173-175such that it can hybridize to said nucleotide sequence shown in SEQ IDNOs: 161-171 and 173-175, thereby forming a stable duplex.

Another object of the invention is a purified, isolated, or recombinantnucleic acid encoding a THAP-2 to THAP11 or THAP-0 polypeptidecomprising, consisting essentially of, or consisting of an amino acidsequence selected from the group consisting of SEQ ID NOs: 4-14, 17-21,23-40, 42-56, 58-98, 100-114 or fragments thereof, wherein the isolatednucleic acid molecule encodes a THAP domain or a THAP-2 to THAP11 orTHAP-0 target binding region. Preferably said target binding region is aprotein binding region, preferably a PAR-4 binding region, or preferablysaid target binding region is a DNA binding region. For example, thepurified, isolated or recombinant nucleic acid may comprise a genomicDNA or fragment thereof which encodes a polypeptide having a sequenceselected from the group consisting of SEQ ID NOs: 4-14, 17-21, 23-40,42-56, 58-98, 100-114 or a fragment thereof. The purified, isolated orrecombinant nucleic acid may alternatively comprise a cDNA consistingof, consisting essentially of, or comprising a sequence selected fromthe group consisting of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98,100-114 or fragments thereof, wherein the isolated nucleic acid moleculeencodes a THAP domain or a THAP-2 to THAP11 or THAP-0 target bindingregion. In preferred embodiments, a THAP-2 to THAP11 or THAP-0 nucleicacid encodes a THAP-2 to THAP11 or THAP-0 polypeptide comprising atleast two THAP-2 to THAP11 or THAP-0 functional domains, such as forexample a THAP domain and a THAP-2 to THAP11 or THAP-0 target bindingregion.

Particularly preferred nucleic acids of the invention include isolated,purified, or recombinant THAP-2 to THAP11 or THAP-0 nucleic acidscomprising, consisting essentially of, or consisting of a contiguousspan of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 150, 200 or 250 nucleotides of a sequence selected from the groupconsisting of nucleotide positions coding for the relevant amino acidsas given in the SEQ ID NO: 161-171 and 173-175.

In further preferred embodiments, a THAP-2 to THAP11 or THAP-0 nucleicacid comprises a nucleotide sequence encoding a THAP domain having theconsensus amino acid sequence of the formula of SEQ ID NOs: 1-2. ATHAP-2 to THAP11 or THAP-0 nucleic acid may also encode a THAP domainwherein at least about 95%, 90%, 85%, 50-80%, preferably at least about60-70%, more preferably at least about 65% of the amino acid residuesare identical or similar amino acids-to the THAP consensus domain (SEQID NOs: 1-2). The present invention also embodies isolated, purified,and recombinant polynucleotides which encode a polypeptide comprising acontiguous span of at least 6 amino acids, preferably at least 8 or 10amino acids, more preferably at least 15, 25, 30, 35, 40, 45, 50, 60,70, 80 or 90 amino acids of SEQ ID NOs: 1-2.

The nucleotide sequence determined from the cloning of the THAP-2 toTHAP11 or THAP-0 genes allows for the generation of probes and primersdesigned for use in identifying and/or cloning other THAP familymembers, particularly sequences related to THAP-2 to THAP11 or THAP-0(e.g. sharing the novel functional domains), as well as THAP-2 to THAP11or THAP-0 homologues from other species.

A nucleic acid fragment encoding a biologically active portion of aTHAP-2 to THAP11 or THAP-0 protein can be prepared by isolating aportion of a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 161-171 and 173-175, which encodes a polypeptide having aTHAP-2 to THAP11 or THAP-0 biological activity (the biologicalactivities of the THAP-family proteins described herein), expressing theencoded portion of the THAP-2 to THAP11 or THAP-0 protein (e.g., byrecombinant expression in vitro or in vivo) and assessing the activityof the encoded portion of the THAP-2 to THAP11 or THAP-0 protein.

The invention further encompasses nucleic acid molecules that differfrom the THAP-2 to THAP11 or THAP-0 nucleotide sequences of theinvention due to degeneracy of the genetic code and encode the sameTHAP-2 to THAP11 or THAP-0 protein, or fragment thereof, of theinvention.

In addition to the THAP-2 to THAP11 or THAP-0 nucleotide sequencesdescribed above, it will be appreciated by those skilled in the art thatDNA sequence polymorphisms that lead to changes in the amino acidsequences of the respective THAP-2 to THAP11 or THAP-0 protein may existwithin a population (e.g., the human population). Such geneticpolymorphism may exist among individuals within a population due tonatural allelic variation. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of a particularTHAP-2 to THAP11 or THAP-0 gene.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the THAP-2 to THAP11 or THAP-0 nucleic acids of theinvention can be isolated based on their homology to the THAP-2 toTHAP11 or THAP-0 nucleic acids disclosed herein using the cDNAsdisclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions.

Probes based on the THAP-2 to THAP11 or THAP-0 nucleotide sequences canbe used to detect transcripts or genomic sequences encoding the same orhomologous proteins. In preferred embodiments, the probe furthercomprises a label group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress a THAP-2 to THAP11 orTHAP-0 protein, such as by measuring a level of a THAP-2 to THAP11 orTHAP-0-encoding nucleic acid in a sample of cells from a subject e.g.,detecting THAP-2 to THAP11 or THAP-0 mRNA levels or determining whethera genomic THAP-2 to THAP11 or THAP-0 gene has been mutated or deleted.

THAP-2 to THAP11 and THAP-0 Polypeptides

The term “THAP-2 to THAP11 or THAP-0 polypeptides” is used herein toembrace all of the proteins and polypeptides of the present inventionrelating to THAP-2, THAP-3, THAP-4, THAP-5, THAP-6, THAP-7, THAP-8,THAP-9, THAP10, THAP11 and THAP-0. Also forming part of the inventionare polypeptides encoded by the polynucleotides of the invention, aswell as fusion polypeptides comprising such polypeptides. The inventionembodies THAP-2 to THAP11 or THAP-0 proteins from humans, includingisolated or purified THAP-2 to THAP11 or THAP-0 proteins consisting of,consisting essentially of, or comprising a sequence selected from thegroup consisting of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 and100-114.

The invention concerns the polypeptide encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 161-171, 172-175 and acomplementary sequence thereof and a fragment thereof.

The present invention embodies isolated, purified, and recombinantpolypeptides comprising a contiguous span of at least 6 amino acids,preferably at least 8 to 10 amino acids, more preferably at least 12,15, 20, 25, 30, 40, 50, 100, 150, 200, 300 or 500 amino acids, to theextent that said span is consistent with the particular SEQ ID NO:, of asequence selected from the group consisting of SEQ ID NOs: 4-14, 17-21,23-40, 42-56, 58-98 and 100-114. In other preferred embodiments thecontiguous stretch of amino acids comprises the site of a mutation orfunctional mutation, including a deletion, addition, swap or truncationof the amino acids in the THAP-2 to THAP11 or THAP-0 protein sequence.

One aspect of the invention pertains to isolated THAP-2 to THAP11 andTHAP-0 proteins, and biologically active portions thereof, as well aspolypeptide fragments suitable for use as immunogens to raiseanti-THAP-2 to THAP11 or THAP-0 antibodies. In one embodiment, nativeTHAP-2 to THAP11 or THAP-0 proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, THAP-2 to THAP11 orTHAP-0 proteins are produced by recombinant DNA techniques. Alternativeto recombinant expression, a THAP-2 to THAP11 or THAP-0 protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

Biologically active portions of a THAP-2 to THAP11 or THAP-0 proteininclude peptides comprising amino acid sequences sufficiently homologousto or derived from the amino acid sequence of the THAP-2 to THAP11 orTHAP-0 protein, e.g., an amino acid sequence shown in SEQ ID NOs: 4-14,17-21, 23-40, 42-56, 58-98 or 100-114, which include less amino acidsthan the respective full length THAP-2 to THAP11 or THAP-0 protein, andexhibit at least one activity of the THAP-2 to THAP11 or THAP-0 protein.The present invention also embodies isolated, purified, and recombinantportions or fragments of a THAP-2 to THAP11 or THAP-0 polypeptidecomprising a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 100, 150, 200, 300 or 500 amino acids, to the extent that saidspan is consistent with the particular SEQ ID NO, of a sequence selectedfrom the group consisting of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56,58-98 and 100-114. Also encompassed are THAP-2 to THAP11 or THAP-0polypeptides which comprise between 10 and 20, between 20 and 50,between 30 and 60, between 50 and 100, or between 100 and 200 aminoacids of a sequence selected from the group consisting of SEQ ID NOs:4-14, 17-21, 23-40, 42-56, 58-98 and 100-114. In other preferredembodiments the contiguous stretch of amino acids comprises the site ofa mutation or functional mutation, including a deletion, addition, swapor truncation of the amino acids in the THAP-2 to THAP11 or THAP-0protein sequence.

A biologically active THAP-2 to THAP11 or THAP-0 protein may, forexample, comprise at least 1, 2, 3, 5, 10, 20 or 30 amino acid changesfrom the sequence of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 or100-114, or may encode a biologically active THAP-2 to THAP11 or THAP-0protein comprising at least 1%, 2%, 3%, 5%, 8%, 10% or 15% changes inamino acids from the sequence of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56,58-98 or 100-114.

In a preferred embodiment, the THAP-2 protein comprises, consistsessentially of, or consists of a THAP-2 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 89 shown in SEQ IDNO: 4, or fragments or variants thereof. The invention also concerns thepolypeptide encoded by the THAP-2 nucleotide sequences of the invention,or a complementary sequence thereof or a fragment thereof. The presentinvention thus also embodies isolated, purified, and recombinantpolypeptides comprising, consisting essentially of or consisting of acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 70, 80or 89 amino acids of a sequence comprising amino acid positions 1 to 89of SEQ ID NO: 4. In another aspect, a THAP-2 polypeptide may comprise aTHAP domain wherein at least about 95%, 90%, 85%, 50-80%, preferably atleast about 60-70%, more preferably at least about 65% of the amino acidresidues are identical or similar amino acids-to the THAP domainconsensus domain (SEQ ID NOs: 1-2). Also encompassed by the presentinvention are isolated, purified, nucleic acids encoding a THAP-2polypeptide comprising, consisting essentially of, or consisting of aTHAP domain at amino acid positions 1 to 89 shown in SEQ ID NO: 4, orfragments or variants thereof. Preferably, said THAP-2 polypeptidecomprises a PAR-4 binding domain and/or a DNA binding domain.

In a preferred embodiment, the THAP-3 protein comprises, consistsessentially of, or consists of a THAP-3 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 89 shown in SEQ IDNO: 5, or fragments or variants thereof. The invention also concerns thepolypeptide encoded by the THAP-3 nucleotide sequences of the invention,or a complementary sequence thereof or a fragment thereof. The presentinvention thus also embodies isolated, purified, and recombinantpolypeptides comprising, consisting essentially of or consisting of acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 70, 80or 89 amino acids of a sequence comprising amino acid positions 1 to 89of SEQ ID NO: 5. In another aspect, a THAP-3 polypeptide may comprise aTHAP domain wherein at least about 95%, 90%, 85%, 50-80%, preferably atleast about 60-70%, more preferably at least about 65% of the amino acidresidues are identical or similar amino acids-to the THAP domainconsensus domain (SEQ ID NOs: 1-2). Also encompassed by the presentinvention are isolated, purified, nucleic acids encoding a THAP-3polypeptide comprising, consisting essentially of, or consisting of aTHAP domain at amino acid positions 1 to 89 shown in SEQ ID NO: 5, orfragments or variants thereof. Preferably, said THAP-3 polypeptidecomprises a PAR-4 binding domain and/or a DNA binding domain.

In a preferred embodiment, the THAP-4 protein comprises, consistsessentially of, or consists of a THAP-4 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 6, or fragments or variants thereof. The invention also concerns thepolypeptide encoded by the THAP-4 nucleotide sequences of the invention,or a complementary sequence thereof or a fragment thereof. The presentinvention thus also embodies isolated, purified, and recombinantpolypeptides comprising, consisting essentially of or consisting of acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 70, 80or 90 amino acids of a sequence comprising amino acid positions 1 to 90of SEQ ID NO: 6. In another aspect, a THAP-4 polypeptide may comprise aTHAP domain wherein at least about 95%, 90%, 85%, 50-80%, preferably atleast about 60-70%, more preferably at least about 65% of the amino acidresidues are identical or similar amino acids-to the THAP domainconsensus domain (SEQ ID NOs: 1-2). Also encompassed by the presentinvention are isolated, purified, nucleic acids encoding a THAP-4polypeptide comprising, consisting essentially of, or consisting of aTHAP domain at amino acid positions 1 to 90 shown in SEQ ID NO: 6, orfragments or variants thereof.

In a preferred embodiment, the THAP-5 protein comprises, consistsessentially of, or consists of a THAP-5 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 7, or fragments or variants thereof. The invention also concerns thepolypeptide encoded by the THAP-5 nucleotide sequences of the invention,or a complementary sequence thereof or a fragment thereof. The presentinvention thus also embodies isolated, purified, and recombinantpolypeptides comprising, consisting essentially of or consisting of acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 70, 80or 90 amino acids of a sequence comprising amino acid positions 1 to 90of SEQ ID NO: 7. In another aspect, a THAP-5 polypeptide may comprise aTHAP domain wherein at least about 95%, 90%, 85%, 50-80%, preferably atleast about 60-70%, more preferably at least about 65% of the amino acidresidues are identical or similar amino acids-to the THAP domainconsensus domain (SEQ ID NOs: 1-2). Also encompassed by the presentinvention are isolated, purified, nucleic acids encoding a THAP-5polypeptide comprising, consisting essentially of, or consisting of aTHAP domain at amino acid positions 1 to 90 shown in SEQ ID NO: 7, orfragments or variants thereof.

In a preferred embodiment, the THAP-6 protein comprises, consistsessentially of, or consists of a THAP-6 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 8, or fragments or variants thereof. The invention also concerns thepolypeptide encoded by the THAP-6 nucleotide sequences of the invention,or a complementary sequence thereof or a fragment thereof. The presentinvention thus also embodies isolated, purified, and recombinantpolypeptides comprising, consisting essentially of or consisting of acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 70, 80or 90 amino acids of a sequence comprising amino acid positions 1 to 90of SEQ ID NO: 8. In another aspect, a THAP-6 polypeptide may comprise aTHAP domain wherein at least about 95%, 90%, 85%, 50-80%, preferably atleast about 60-70%, more preferably at least about 65% of the amino acidresidues are identical or similar amino acids-to the THAP domainconsensus domain (SEQ ID NOs: 1-2). Also encompassed by the presentinvention are isolated, purified, nucleic acids encoding a THAP-6polypeptide comprising, consisting essentially of, or consisting of aTHAP domain at amino acid positions 1 to 90 shown in SEQ ID NO: 8, orfragments or variants thereof.

In a preferred embodiment, the THAP-7 protein comprises, consistsessentially of, or consists of a THAP-7 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 9, or fragments or variants thereof. The invention also concerns thepolypeptide encoded by the THAP-7 nucleotide sequences of the invention,or a complementary sequence thereof or a fragment thereof. The presentinvention thus also embodies isolated, purified, and recombinantpolypeptides comprising, consisting essentially of or consisting of acontiguous span of at least 6. amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 70, 80or 90 amino acids of a sequence comprising amino acid positions 1 to 90of SEQ ID NO: 9. In another aspect, a THAP-7 polypeptide may comprise aTHAP domain wherein at least about 95%, 90%, 85%, 50-80%, preferably atleast about 60-70%, more preferably at least about 65% of the amino acidresidues are identical or similar amino acids-to the THAP domainconsensus domain (SEQ ID NOs: 1-2). Also encompassed by the presentinvention are isolated, purified, nucleic acids encoding a THAP-7polypeptide comprising, consisting essentially of, or consisting of aTHAP domain at amino acid positions 1 to 90 shown in SEQ ID NO: 9, orfragments or variants thereof.

In a preferred embodiment, the THAP-8 protein comprises, consistsessentially of, or consists of a THAP-8 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 92 shown in SEQ IDNO: 10, or fragments or variants thereof. The invention also concernsthe polypeptide encoded by the THAP-8 nucleotide sequences of theinvention, or a complementary sequence thereof or a fragment thereof.The present invention thus also embodies isolated, purified, andrecombinant polypeptides comprising, consisting essentially of orconsisting of a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 70, 80 or 90 amino acids of a sequence comprising amino acidpositions 1 to 92 of SEQ ID NO: 10. In another aspect, a THAP-8polypeptide may comprise a THAP domain wherein at least about 95%, 90%,85%, 50-80%, preferably at least about 60-70%, more preferably at leastabout 65% of the amino acid residues are identical or similar aminoacids-to the THAP domain consensus domain (SEQ ID NOs: 1-2). Alsoencompassed by the present invention are isolated, purified, nucleicacids encoding a THAP-8 polypeptide comprising, consisting essentiallyof, or consisting of a THAP domain at amino acid positions 1 to 92 shownin SEQ ID NO: 10, or fragments or variants thereof.

In a preferred embodiment, the THAP-9 protein comprises, consistsessentially of, or consists of a THAP-9 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 92 shown in SEQ IDNO: 11, or fragments or variants thereof. The invention also concernsthe polypeptide encoded by the THAP-9 nucleotide sequences of theinvention, or a complementary sequence thereof or a fragment thereof.The present invention thus also embodies isolated, purified, andrecombinant polypeptides comprising, consisting essentially of orconsisting of a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 70, 80 or 90 amino acids of a sequence comprising amino acidpositions 1 to 92 of SEQ ID NO: 11. In another aspect, a THAP-9polypeptide may comprise a THAP domain wherein at least about 95%, 90%,85%, 50-80%, preferably at least about 60-70%, more preferably at leastabout 65% of the amino acid residues are identical or similar aminoacids-to the THAP domain consensus domain (SEQ ID NOs: 1-2). Alsoencompassed by the present invention are isolated, purified, nucleicacids encoding a THAP-9 polypeptide comprising, consisting essentiallyof, or consisting of a THAP domain at amino acid positions 1 to 92 shownin SEQ ID NO: 11, or fragments or variants thereof.

In a preferred embodiment, the THAP10 protein comprises, consistsessentially of, or consists of a THAP10 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 12, or fragments or variants thereof. The invention also concernsthe polypeptide encoded by the THAP 10 nucleotide sequences of theinvention, or a complementary sequence thereof or a fragment thereof.The present invention thus also embodies isolated, purified, andrecombinant polypeptides comprising, consisting essentially of orconsisting of a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 70, 80 or 90 amino acids of a sequence comprising amino acidpositions 1 to 90 of SEQ ID NO: 12. In another aspect, a THAP10polypeptide may comprise a THAP domain wherein at least about 95%, 90%,85%, 50-80%, preferably at least about 60-70%, more preferably at leastabout 65% of the amino acid residues are identical or similar aminoacids-to the THAP domain consensus domain (SEQ ID NOs: 1-2). Alsoencompassed by the present invention are isolated, purified, nucleicacids encoding a THAP10 polypeptide comprising, consisting essentiallyof, or consisting of a THAP domain at amino acid positions 1 to 90 shownin SEQ ID NO: 12, or fragments or variants thereof.

In a preferred embodiment, the THAP11 protein comprises, consistsessentially of, or consists of a THAP11 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 13, or fragments or variants thereof. The invention also concernsthe polypeptide encoded by the THAP11 nucleotide sequences of theinvention, or a complementary sequence thereof or a fragment thereof.The present invention thus also embodies isolated, purified, andrecombinant polypeptides comprising, consisting essentially of orconsisting of a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 70, 80 or 90 amino acids of a sequence comprising amino acidpositions 1 to 90 of SEQ ID NO: 13. In another aspect, a THAP11polypeptide may comprise a THAP domain wherein at least about 95%, 90%,85%, 50-80%, preferably at least about 60-70%, more preferably at leastabout 65% of the amino acid residues are identical or similar aminoacids-to the THAP domain consensus domain (SEQ ID NOs: 1-2). Alsoencompassed by the present invention are isolated, purified, nucleicacids encoding a THAP11 polypeptide comprising, consisting essentiallyof, or consisting of a THAP domain at amino acid positions 1 to 90 shownin SEQ ID NO: 13, or fragments or variants thereof.

In a preferred embodiment, the THAP-0 protein comprises, consistsessentially of, or consists of a THAP-0 THAP domain, preferably havingthe amino acid sequence of amino acid positions 1 to 90 shown in SEQ IDNO: 14, or fragments or variants thereof. The invention also concernsthe polypeptide encoded by the THAP-0 nucleotide sequences of theinvention, or a complementary sequence thereof or a fragment thereof.The present invention thus also embodies isolated, purified, andrecombinant polypeptides comprising, consisting essentially of orconsisting of a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 70, 80 or 90 amino acids of a sequence comprising amino acidpositions 1 to 90 of SEQ ID NO: 14. In another aspect, a THAP-0polypeptide may comprise a THAP domain wherein at least about 95%, 90%,85%, 50-80%, preferably at least about 60-70%, more preferably at leastabout 65% of the amino acid residues are identical or similar aminoacids-to the THAP domain consensus domain (SEQ ID NOs: 1-2). Alsoencompassed by the present invention are isolated, purified, nucleicacids encoding a THAP-0 polypeptide comprising, consisting essentiallyof, or consisting of a THAP domain at amino acid positions 1 to 90 shownin SEQ ID NO: 14, or fragments or variants thereof.

In other embodiments, the THAP-2 to THAP11 or THAP-0 protein issubstantially homologous to the sequences of SEQ ID NOs: 4-14, 17-21,23-40, 42-56, 58-98 or 100-114 and retains the functional activity ofthe THAP-2 to THAP11 or THAP-0 protein, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedfurther herein. Accordingly, in another embodiment, the THAP-2 to THAP11or THAP-0 protein is a protein which comprises an amino acid sequencethat shares more than about 60% but less than 100% homology with theamino acid sequence of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 or100-114 and retains the functional activity of the THAP-2 to THAP11 orTHAP-0 proteins of SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 or100-114, respectively. Preferably, the protein is at least about 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 99.8%homologous to SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 or 100-114,but is not identical to SEQ ID NOs: 4-14, 17-21, 23-40, 42-56, 58-98 or100-114. Preferably the THAP-2 to THAP11 or THAP-0 is less thanidentical (e.g. 100% identity) to a naturally occurring THAP-2 to THAP11or THAP-0. Percent homology can be determined as further detailed above.

Assessing Polypeptides, Methods for Obtaining Variant Nucleic Acids andPolypeptides

It will be appreciated that by characterizing the function ofTHAP-family polypeptides, the invention further provides methods oftesting the activity of, or obtaining, functional fragments and variantsof THAP-family and THAP domain nucleotide sequences involving providinga variant or modified THAP-family or THAP domain nucleic acid andassessing whether a polypeptide encoded thereby displays a THAP-familyactivity of the invention. Encompassed is thus a method of assessing thefunction of a THAP-family or THAP domain polypeptide comprising: (a)providing a THAP family or THAP domain polypeptide, or a biologicallyactive fragment or homologue thereof; and (b) testing said THAP familyor THAP domain polypeptide, or a biologically active fragment orhomologue thereof for a THAP-family activity. Any suitable format may beused, including cell free, cell-based and in vivo formats. For example,said assay may comprise expressing a THAP-family or THAP domain nucleicacid in a host cell, and observing THAP-family activity in said cell. Inanother example, a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof is introduced to acell, and a THAP-family activity is observed. THAP-family activity maybe any activity as described herein, including (1) mediating apoptosisor cell proliferation when expressed or introduced into a cell, mostpreferably inducing or enhancing apoptosis, and/or most preferablyreducing cell proliferation; (2) mediating apoptosis or cellproliferation of an endothelial cell; (3) mediating apoptosis or cellproliferation of a hyperproliferative cell; (4) mediating apoptosis orcell proliferation of a CNS cell, preferably a neuronal or glial cell;or (5) an activity determined in an animal selected from the groupconsisting of mediating, preferably inhibiting angiogenesis, mediating,preferably inhibiting inflammation, inhibition of metastatic potentialof cancerous tissue, reduction of tumor burden, increase in sensitivityto chemotherapy or radiotherapy, killing a cancer cell, inhibition ofthe growth of a cancer cell, or induction of tumor regression.

In addition to naturally-occurring allelic variants of the THAP-familyor THAP domain sequences that may exist in the population, the skilledartisan will appreciate that changes can be introduced by mutation intothe nucleotide sequences of SEQ ID NOs: 160-171, thereby leading tochanges in the amino acid sequence of the encoded THAP-family or THAPdomain proteins, with or without altering the functional ability of theTHAP-family or THAP domain proteins.

Several types of variants are contemplated including 1) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue and such substituted amino acid residuemay or may not be one encoded by the genetic code, or 2) one in whichone or more of the amino acid residues includes a substituent group, or3) one in which the mutated THAP-family or THAP domain polypeptide isfused with another compound, such as a compound to increase thehalf-life of the polypeptide (for example, polyethylene glycol), or 4)one in which the additional amino acids are fused to the mutatedTHAP-family or THAP domain polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the mutatedTHAP-family or THAP domain polypeptide or a preprotein sequence. Suchvariants are deemed to be within the scope of those skilled in the art.

For example, nucleotide substitutions leading to amino acidsubstitutions can be made in the sequences of SEQ ID NOs: 160-175 thatdo not substantially change the biological activity of the protein. Anamino acid residue-can be altered from the wild-type sequence encoding aTHAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof without altering the biological activity.In general, amino acid residues that are conserved among the THAP-familyof THAP domain-containing proteins of the present invention, arepredicted to be less amenable to alteration. Furthermore, additionalconserved amino acid residues may be amino acids that are conservedbetween the THAP-family proteins of the present invention.

In one aspect, the invention pertains to nucleic acid molecules encodingTHAP family or THAP domain polypeptides, or biologically activefragments or homologues thereof that contain changes in amino acidresidues that are not essential for activity. Such THAP-family proteinsdiffer in amino acid sequence from SEQ ID NOs: 1-114 yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 60% homologousto an amino acid sequence selected from the group consisting of SEQ IDNOs: 1-114. Preferably, the protein encoded by the nucleic acid moleculeis at least about 65-70% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-114, more preferably sharingat least about 75-80% identity with an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-114, even more preferably sharingat least about 85%, 90%, 92%, 95%, 97%, 98%, 99% or 99.8% identity withan amino acid sequence selected from the group consisting of SEQ ID NOs:1-114.

In another aspect, the invention pertains to nucleic acid moleculesencoding THAP-family proteins that contain changes in amino acidresidues that result in increased biological activity, or a modifiedbiological activity. In another aspect, the invention pertains tonucleic acid molecules encoding THAP-family proteins that containchanges in amino acid residues that are essential for a THAP-familyactivity. Such THAP-family proteins differ in amino acid sequence fromSEQ ID NOs: 1-114 and display reduced or essentially lack one or moreTHAP-family biological activities. The invention also encompasses a THAPfamily or THAP domain polypeptide, or a biologically active fragment orhomologue thereof which may be useful as dominant negative mutant of aTHAP family or THAP domain polypeptide.

An isolated nucleic acid molecule encoding a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereofhomologous to a protein of any one of SEQ ID NOs: 1-114 can be createdby introducing one or more nucleotide substitutions, additions ordeletions into the nucleotide sequence of SEQ ID NOs: 1-114 such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intoany of SEQ ID NOs: 1-114, by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. For example, conservativeamino acid substitutions may be made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof may be replaced withanother amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a THAP-family or THAP domain codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for THAP-family biological activity to identify mutantsthat retain activity. Following mutagenesis of one of SEQ ID NOs: 1-114,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.

In a preferred embodiment, a mutant THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereofencoded by a THAP family or THAP domain polypeptide, or a biologicallyactive fragment or homologue thereof of THAP domain nucleic acid of theinvention can be assayed for a THAP-family activity in any suitableassay, examples of which are provided herein.

The invention also provides THAP-family or THAP domain chimeric orfusion proteins. As used herein, a THAP-family or THAP domain “chimericprotein” or “fusion protein” comprises a THAP-family or THAP domainpolypeptide of the invention operatively linked, preferably fused inframe, to a non-THAP-family or non-THAP domain polypeptide. In apreferred embodiment, a THAP-family or THAP domain fusion proteincomprises at least one biologically active portion of a THAP-family orTHAP domain protein. In another preferred embodiment, a THAP-familyfusion protein comprises at least two biologically active portions of aTHAP-family protein. For example, in one embodiment, the fusion proteinis a GST-THAP-family fusion protein in which the THAP-family sequencesare fused to the C-terminus of the GST sequences. Such fusion proteinscan facilitate the purification of recombinant THAP-family polypeptides.In another embodiment, the fusion protein is a THAP-family proteincontaining a heterologous signal sequence at its N-terminus, such as forexample to allow for a desired cellular localization in a certain hostcell.

The THAP-family or THAP domain fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject in vivo. Moreover, the THAP-family-fusion or THAP domainproteins of the invention can be used as immunogens to produceanti-THAP-family or anti or THAP domain antibodies in a subject, topurify THAP-family or THAP domain ligands and in screening assays toidentify molecules which inhibit the interaction of THAP-family or THAPdomain with a THAP-family or THAP domain target molecule.

Furthermore, isolated peptidyl portions of the subject THAP-family orTHAP domain proteins can also be obtained by screening peptidesrecombinantly produced from the corresponding fragment of the nucleicacid encoding such peptides. In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. For example, aTHAP-family or THAP domain protein of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of a THAP-familyprotein activity, such as by microinjection assays or in vitro proteinbinding assays. In an illustrative embodiment, peptidyl portions of aTHAP-family protein, such as a THAP domain or a THAP-family targetbinding region (e.g. PAR4 in the case of THAP1, THAP-2 and THAP-3), canbe tested for THAP-family activity by expression as thioredoxin fusionproteins, each of which contains a discrete fragment of the THAP-familyprotein (see, for example, U.S. Pat. Nos. 5,270,181 and 5,292,646; andPCT publication WO94/02502, the disclosures of which are incorporatedherein by reference).

The present invention also pertains to variants of the THAP-family orTHAP domain proteins which function as either THAP-family or THAP domainmimetics or as THAP-family or THAP domain inhibitors. Variants of theTHAP-family or THAP domain proteins can be generated by mutagenesis,e.g., discrete point mutation or truncation of a THAP-family or THAPdomain protein. An agonist of a THAP-family or THAP domain protein canretain substantially the same, or a subset, of the biological activitiesof the naturally occurring form of a THAP-family or THAP domain protein.An antagonist of a THAP-family or THAP domain protein can inhibit one ormore of the activities of the naturally occurring form of theTHAP-family or THAP domain protein by, for example, competitivelyinhibiting the association of a THAP-family or THAP domain protein witha THAP-family target molecule. Thus, specific biological effects can beelicited by treatment with a variant of limited function. In oneembodiment, variants of a THAP-family or THAP domain protein whichfunction as either THAP-family or THAP domain agonists (mimetics) or asTHAP-family or THAP domain antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of aTHAP-family or THAP domain protein for THAP-family or THAP domainprotein agonist or antagonist activity. In one embodiment, a variegatedlibrary of THAP-family variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of THAP-family variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential THAP-family sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of THAP-family sequencestherein. There are a variety of methods which can be used to producelibraries of potential THAP-family variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potentialTHAP-family sequences.

In addition, libraries of fragments of a THAP-family or THAP domainprotein coding sequence can be used to generate a variegated populationof THAP-family or THAP domain fragments for screening and subsequentselection of variants of a THAP-family or THAP domain protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a THAP-family coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the THAP-family protein.

Modified THAP-family or THAP domain proteins can be used for suchpurposes as enhancing therapeutic or prophylactic efficacy, or stability(e.g., ex vivo shelf life and resistance to proteolytic degradation invivo). Such modified peptides, when designed to retain at least oneactivity of the naturally occurring form of the protein, are consideredfunctional equivalents of the THAP-family or THAP domain proteindescribed in more detail herein. Such modified peptide can be produced,for instance, by amino acid substitution, deletion, or addition.

Whether a change in the amino acid sequence of a peptide results in afunctional THAP-family or THAP domain homolog (e.g. functional in thesense that it acts to mimic or antagonize the wild-type form) can bereadily determined by assessing the ability of the variant peptide toproduce a response in cells in a fashion similar to the wild-typeTHAP-family or THAP domain protein or competitively inhibit such aresponse. Peptides in which more than one replacement has taken placecan readily be tested in the same manner.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the presently disclosed THAP-family or THAPdomain proteins, as well as truncation and fragmentation mutants, and isespecially useful for identifying potential variant sequences which arefunctional in binding to a THAP-family- or THAP domain-target proteinbut differ from a wild-type form of the protein by, for example,efficacy, potency and/or intracellular half-life. One purpose forscreening such combinatorial libraries is, for example, to isolate novelTHAP-family or THAP domain homologs which function as either an agonistor an antagonist of the biological activities of the wild-type protein,or alternatively, possess novel activities all together. For example,mutagenesis can give rise to THAP-family homologs which haveintracellular half-lives dramatically different than the correspondingwild-type protein. The altered protein can be rendered either morestable or less stable to proteolytic degradation or other cellularprocess which result in destruction of, or otherwise inactivation of, aTHAP-family protein. Such THAP-family homologs, and the genes whichencode them, can be utilized to alter the envelope of expression for aparticular recombinant THAP-family protein by modulating the half-lifeof the recombinant protein. For instance, a short half-life can giverise to more transient biological effects associated with a particularrecombinant THAP-family protein and, when part of an inducibleexpression system, can allow tighter control of recombinant proteinlevels within a cell. As above, such proteins, and particularly theirrecombinant nucleic acid constructs, can be used in gene therapyprotocols.

In an illustrative embodiment of this method, the amino acid sequencesfor a population of THAP-family homologs or other related proteins arealigned, preferably to promote the highest homology possible. Such apopulation of variants can include, for example, THAP-family homologsfrom one or more species, or THAP-family homologs from the same speciesbut which differ due to mutation. Amino acids which appear at eachposition of the aligned sequences are selected to create a degenerateset of combinatorial sequences. There are many ways by which the libraryof potential THAP-family homologs can be generated from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic genes then be ligated into an appropriate gene for expression.The purpose of a degenerate set of genes is to provide, in one mixture,all of the sequences encoding the desired set of potential THAP-familysequences. The synthesis of degenerate oligonucleotides is well known inthe art (see for example. Narang, S A (1983) Tetrahedron 393; Itakura etal. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules,ed. A G Walton, Amsterdam: Elsevier pp. 273-289; Itakura et al. (1984)Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ikeet al. (1983) Nucleic Acid Res. 11:477. Such techniques have beenemployed in the directed evolution of other proteins (see, for example,Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al.(1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815). The disclosures of the above references areincorporated herein by reference in their entireties.

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library, particularly where no other naturally occurringhomologs have yet been sequenced. For example, THAP-family homologs(both agonist and antagonist forms) can be generated and isolated from alibrary by screening using, for example, alanine scanning mutagenesisand the like (Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al.(1994) J Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene137:109-118; Grodberg et al. (1993) Eur. J Biochem. 218:597-601;Nagashima et al. (1993) J Biol. Chem. 268:2888-2892; Lowman et al.(1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science244:1081-1085), by linker scanning mutagenesis (Gustin et al. (1993)Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644 2652;McKnight et al. (1982) Science 232:316); by saturation mutagenesis(Meyers et al. (1986) Science 232:613); by PCR mutagenesis (Leung et al.(1989) Method Cell Mol Biol 1: 1-19); or by random mutagenesis (Milleret al. (1992) A Short Course in Bacterial Genetics, CSHL Press, ColdSpring Harbor, N.Y.; and Greener et al. (1994) Strategies in Mol Biol7:32-34, the disclosures of which are incorporated herein by referencein their entireties).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, as well asfor screening cDNA libraries for gene products having a certainproperty. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of THAP-family proteins. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected.

Each of the illustrative assays described below are amenable to highthrough-put analysis as necessary to screen large numbers of degenerateTHAP-family or THAP domain sequences created by combinatorialmutagenesis techniques. In one screening assay, the candidate geneproducts are displayed on the surface of a cell or viral particle, andthe ability of particular cells or viral particles to bind a THAP-familytarget molecule (protein or DNA) via this gene product is detected in a“panning assay”. For instance, the gene library can be cloned into thegene for a surface membrane protein of a bacterial cell, and theresulting fusion protein detected by panning (Ladner et al., WO88/06630; Fuchs et al. (1991) BiolTechnology 9:1370-1371, and Goward etal. (1992) TIBS 18:136 140). In a similar fashion, fluorescently labeledTHAP-family target can be used to score for potentially functionalTHAP-family homologs. Cells can be visually inspected and separatedunder a fluorescence microscope, or, where the morphology of the cellpermits, separated by a fluorescence-activated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, a large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and fl are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:44574461, the disclosures of which are incorporated herein by reference intheir entireties). In an illustrative embodiment, the recombinant phageantibody system (RPAS, Pharmacia Catalog number 27-9400-01) can beeasily modified for use in expressing THAP-family combinatoriallibraries, and the THAP-family phage library can be panned onimmobilized THAP family target molecule (glutathione immobilizedTHAP-family target-GST fusion proteins or immobilized DNA). Successiverounds of phage amplification and panning can greatly enrich forTHAP-family homologs which retain an ability to bind a THAP-familytarget and which can subsequently be screened further for biologicalactivities in automated assays, in order to distinguish between agonistsand antagonists.

The invention also provides for identification and reduction tofunctional minimal size of the THAP-family domains, particularly a THAPdomain of the subject THAP-family to generate mimetics, e.g. peptide ornon-peptide agents, which are able to disrupt binding of a polypeptideof the present invention with a THAP-family target molecule (protein orDNA). Thus, such mutagenic techniques as described above are also usefulto map the determinants of THAP-family proteins which participate inprotein-protein or protein-DNA interactions involved in, for example,binding to a THAP-family or THAP domain target protein or DNA. Toillustrate, the critical residues of a THAP-family protein which areinvolved in molecular recognition of the THAP-family target can bedetermined and used to generate THAP-family target-13P-derivedpeptidomimetics that competitively inhibit binding of the THAP-familyprotein to the THAP-family target. By employing, for example, scanningmutagenesis to map the amino acid residues of a particular THAP-familyprotein involved in binding a THAP-family target, peptidomimeticcompounds can be generated which mimic those residues in binding to aTHAP-family target, and which, by inhibiting binding of the THAP-familyprotein to the THAP-family target molecule, can interfere with thefunction of a THAP-family protein in transcriptional regulation of oneor more genes. For instance, non hydrolyzable peptide analogs of suchresidues can be generated using retro-inverse peptides (e.g., see U.S.Pat. Nos. 5,116,947 and 5,219,089; and Pallai et al. (1983) Int J PeptProtein Res 21:84-92), benzodiazepine (e.g., see Freidinger et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. inPeptides.-Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey etal. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides(Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), P-turndipeptide cores (Nagai et al. (1985) Tetrahedron Left 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1: 123 1), and P-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71, the disclosures of whichare incorporated herein by reference in their entireties).

An isolated THAP-family or THAP domain protein, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindTHAP-family or THAP domain proteins using standard techniques forpolyclonal and monoclonal antibody preparation. A full-lengthTHAP-family protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of THAP-family or THAP domainproteins for use as immunogens. Any fragment of the THAP-family or THAPdomain protein which contains at least one antigenic determinant may beused to generate antibodies. The antigenic peptide of a THAP-family orTHAP domain protein comprises at least 8 amino acid residues of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-114and encompasses an epitope of a THAP-family or THAP domain protein suchthat an antibody raised against the peptide forms a specific immunecomplex with a THAP-family or THAP domain protein. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Preferred epitopes encompassed by the antigenic peptide are regions of aTHAP-family or THAP domain protein that are located on the surface ofthe protein, e.g., hydrophilic regions.

A THAP-family or THAP domain protein immunogen typically is used toprepare antibodies by immunizing a suitable subject, (e.g., rabbit,goat, mouse or other mammal) with the immunogen. An appropriateimmunogenic preparation can contain, for example, recombinantlyexpressed THAP-family or THAP domain protein or a chemically synthesizedTHAP-family or THAP domain polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic THAP-family or THAP domain protein preparationinduces a polyclonal anti-THAP-family or THAP domain protein antibodyresponse.

The invention concerns antibody compositions, either polyclonal ormonoclonal, capable of selectively binding, or selectively bind to anepitope-containing a polypeptide comprising a contiguous span of atleast 6 amino acids, preferably at least 8 to 10 amino acids, morepreferably at least 12, 15, 20, 25, 30, 40, 50, 100, or more than 100amino acids of an amino acid sequence selected from the group consistingof amino acid positions 1 to approximately 90 of SEQ ID NOs: 1-114. Theinvention also concerns a purified or isolated antibody capable ofspecifically binding to a mutated THAP-family or THAP domain protein orto a fragment or variant thereof comprising an epitope of the mutatedTHAP-family or THAP domain protein.

Oligomeric Forms of THAP1

Certain embodiments of the present invention encompass THAP1polypeptides in the form of oligomers, such as dimers, trimers, orhigher oligomers. Oligomers may be formed by disulfide bonds betweencysteine residues on different THAP1 polypeptides, for example. In otherembodiments, oligomers comprise from two to four THAP1 polypeptidesjoined by covalent or non-covalent interactions between peptide moietiesfused to the THAP1 polypeptides. Such peptide moieties may be peptidelinkers (spacers), or peptides that have the property of promotingoligomerization. Leucine zippers and certain polypeptides derived fromantibodies are among the peptides that can promote oligomerization ofTHAP1 polypeptides attached thereto. DNA sequences encoding THAP1oligomers, or fusion proteins that are components of such oligomers, areprovided herein.

In one embodiment of the invention, oligomeric THAP1 may comprise two ormore THAP1 polypeptides joined through peptide linkers. Examples includethose peptide linkers described in U.S. Pat. No. 5,073,627, thedisclosure of which is incorporated herein by reference in its entirety.Fusion proteins comprising multiple THAP1 polypeptides separated bypeptide linkers may be produced using conventional recombinant DNAtechnology.

Another method for preparing THAP1 oligomers involves use of a leucinezipper. Leucine zipper domains are peptides that promote oligomerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing THAP1 oligomers are thosedescribed International Publication WO 94/10308, the disclosure of whichis incorporated herein by reference in its entirety. Recombinant fusionproteins comprising a THAP1 polypeptide fused to a peptide thatdimerizes or trimerizes in solution are expressed in suitable hostcells, and the resulting soluble oligomeric THAP1 is recovered from theculture supernatant.

In some embodiments of the invention, a THAP1 dimer is created by fusingTHAP1 to an Fc region polypeptide derived from an antibody, in a mannerthat does not substantially affect the binding of THAP1 to the chemokineSLC/CCL21. Preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including Fc region) has been described, e.g., by Ashkenazi et al.(1991) PNAS 88:10535, Byrn et al. (1990) Nature 344:667, and Hollenbaughand Aruffo “Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Supp. 4, pages 10.19.1-10.19-11, 1992, thedisclosures of which are incorporated herein by reference in theirentireties. The THAP1/Fc fusion proteins are allowed to assemble muchlike antibody molecules, whereupon interchain disulfide bonds formbetween Fc polypeptides, yielding divalent THAP1. Similar fusionproteins of TNF receptors and Fc (see for example Moreland et al. (1997)N. Engl. J. Med. 337(3):141-147; van der Poll et al. (1997) Blood89(10):3727-3734; and Ammann et al. (1997) J. Clin. Invest.99(7):1699-1703) have been used successfully for treating rheumatoidarthritis. Soluble derivatives have also been made of cell surfaceglycoproteins in the immunoglobulin gene superfamily consisting of anextracellular domain of the cell surface glycoprotein fused to animmunoglobulin constant (Fc) region (see e.g., Capon, D. J. et al.(1989) Nature 337:525-531 and Capon U.S. Pat. Nos. 5,116,964 and5,428,130 [CD4-IgG1 constructs]; Linsley, P. S. et al. (1991) J. Exp.Med. 173:721-730 [a CD28-IgG1 construct and a B7-1-IgG1 construct]; andLinsley, P. S. et al. (1991) J. Exp. Med. 174:561-569 and U.S. Pat. No.5,434,131 [a CTLA4-IgG1], the disclosures of which are incorporatedherein by reference in their entirety). Such fusion proteins have provenuseful for modulating receptor-ligand interactions.

Some embodiments relate to THAP-immunoglobulin fusion proteins and THAPSLC-binding domain fusions with immunoglobulin molecules or fragmentsthereof. Such fusions can be produced using standard methods, forexample, by creating an expression vector encoding the SLC/CCL21chemokine-binding protein THAP1 fused to the antibody polypeptide andinserting the vector into a suitable host cell. One suitable Fcpolypeptide is the native Fc region polypeptide derived from a humanIgG1, which is described in International Publication WO 93/10151, thedisclosure of which is incorporated herein by reference in its entirety.Another useful Fc polypeptide is the Fc mutein described in U.S. Pat.No. 5,457,035, the disclosure of which is incorporated herein byreference in its entirety. The amino acid sequence of the mutein isidentical to that of the native Fc sequence presented in InternationalPublication WO 93/10151, the disclosure of which is incorporated hereinby reference in its entirety, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. This mutein Fc exhibitsreduced affinity for immunoglobulin receptors.

SLC-binding fragments of human THAP1, rather than the full protein, canalso be employed in methods of the invention. Fragments may be lessimmunogenic than the corresponding full-length proteins. The ability ofa fragment to bind chemokine SLC can be determined using a standardassay. Fragments can be prepared by any of a number of conventionalmethods. For example, a desired DNA sequence can be synthesizedchemically or produced by restriction endonuclease digestion of a fulllength cloned DNA sequence and isolated by electrophoresis on agarosegels. Linkers containing restriction endonuclease cleavage sites can beemployed to insert the desired DNA fragment into an expression vector,or the fragment can be digested at naturally-present cleavage sites. Thepolymerase chain reaction (PCR) can also be employed to isolate a DNAsequence encoding a desired protein fragment. Oligonucleotides thatdefine the termini of the desired fragment are used as 5′ and 3′ primersin the PCR procedure. Additionally, known mutagenesis techniques can beused to insert a stop codon at a desired point, e.g., immediatelydownstream of the codon for the last amino acid of the desired fragment.

In other embodiments, THAP1 or a biologically active fragment thereof,for example, an SLC-binding domain of THAP1 may be substituted for thevariable portion of an antibody heavy or light chain. If fusion proteinsare made with both heavy and light chains of an antibody, it is possibleto form a THAP1 oligomer with at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or more than nine THAP1 polypeptides.

In some embodiments of the present invention, THAP-SLC binding can beprovided to decrease the biological availability of SLC or otherwisedisrupt the activity of SLC. For example, THAP-family polypeptides,SLC-binding domains of THAP-family polypeptides, THAP oligomers, andSLC-binding domain-THAP1-immunoglobulin fusion proteins of the inventioncan be used to interact with SLC thereby preventing it from performingits normal biological role. In some embodiments, the entire THAP1polypeptide (SEQ ID NO: 3) can be used to bind to SLC. In otherembodiments, fragments of THAP1, such as the SLC-binding domain of theTHAP1-(amino acids 143-213 of SEQ ID NO: 3) can used to bind to SLC.Such fragments can be from at least 8, at least 10, at least 12, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70, at least 80, at least 90, at least 100, at least 110, at least120, at least 130, at least 140, at least 150, at least 160, at least170, at least 180, at least 190, at least 200, at least 210 or at least213 consecutive amino acids of SEQ ID NO: 3. In some embodiments,fragments can be from at least 8, at least 10, at least 12, at least 15,at least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55, at least 60, at least 65 or at least70 consecutive amino acids of (amino acids 143-213 of SEQ ID NO: 3).THAP-family polypeptides that may be capable of binding SLC, for exampleTHAP2-11 and THAP0 or biologically active fragments thereof can also beused to bind to SLC so as to decrease its biological availability orotherwise disrupt the activity of this chemokine.

In some embodiments, a plurality of THAP-family proteins, such as afusion of two or more THAP1 proteins or fragments thereof which comprisean SLC-binding domain (amino acids 143-213 of SEQ ID NO: 3) can be usedto bind SLC. For example, oligomers comprising THAP1 fragments of a sizeof at least 8, at least 10, at least 12, at least 15, at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 55, at least 60, at least 65 or at least 70 consecutiveamino acids of SEQ ID NO: 3 (amino acids 143-213) can be generated.Amino acid fragments which make up the THAP oligomer may be of the sameor different lengths. In some embodiments, the entire THAP1 protein orbiologically active portions thereof may be fused together to form anoligomer capable of binding to SLC. THAP-family polypeptides that may becapable of binding SLC, for example THAP2-11 and THAP0, the THAP-familypolypeptides of SEQ ID NOs: 16-114 or biologically active fragmentsthereof can also be used to create oligomers which bind to SLC so as todecrease its biological availability or otherwise disrupt the activityof this chemokine.

According to another embodiment of the present invention, THAP-familyproteins, such as THAP1 or portion of THAP1 which comprise an SLCbinding domain (amino acids 143-213 of SEQ ID NO: 3), may be fused to animmunoglubulin or portion thereof. The portion may be an entireimmunoglobulin, such as IgG, IgM, IgA or IgE. Additionally, portions ofimmunoglobulins, such as an Fc domain of the immunoglobulin, can befused to a THAP-family polypeptide, such as THAP1, fragments thereof oroligomers thereof. Fragments of THAP1 can be, for example, at least 8,at least 10, at least 12, at least 15, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, at least 50, at least55, at least 60, at least 65 or at least 70 consecutive amino acids ofSEQ ID NO: 3 (amino acids 143-213). In some embodiments, THAP-familypolypeptides that may be capable of binding SLC, for example THAP2-11and THAP0, the THAP-family polypeptides of SEQ ID NOs: 16-114 orbiologically active fragments thereof can also be used to formimmunglobulin fusion that bind to SLC so as to decrease its biologicalavailability or otherwise disrupt the activity of this chemokine.

In accordance with another aspect of the invention, THAP-familypolypeptides, SLC-binding domains of THAP-family polypeptides, THAPoligomers, and SLC-binding domain-THAP1-immunoglobulin fusion proteinsof the invention can be incorporated into pharmaceutical compositions.Such pharmaceutical compositions can be used to decrease thebioavailability and functionality of SLC. For example, THAP-familypolypeptides, SLC-binding domains of THAP-family polypeptides, THAPoligomers, and SLC-binding domain-THAP1-immunoglobulin fusion proteinsof the present invention can be administered to a subject to inhibit aninteraction between SLC and its receptor, such as CCR7, on the surfaceof cells, to thereby suppress SLC-mediated responses. The inhibition ofchemokine SLC may be useful therapeutically for both the treatment ofinflammatory or proliferative disorders, as well as modulating (e.g.,promoting or inhibiting) cell differentiation, cell proliferation,and/or cell death.

In an additional embodiment of the present invention, the THAP-familypolypeptides, SLC-binding domains of THAP-family polypeptides, THAPoligomers, and SLC- binding domain-THAP1-immunoglobulin fusion proteinsof the present invention can be used to detect the presence of SLC in abiological sample and in screening assays to identify molecules whichinhibit the interaction of THAP1 with SLC. Such screening assays aresimilar to those described below for PAR4-THAP interactions.

Certain aspects of the present invention related to a method ofidentifying a test compound that modulates THAP-mediated activites. Insome cases the THAP-mediated acitivity is SLC-binding. Test compoundswhich affect THAP-SLC binding can be identified using a screening methodwherein a THAP-family polypeptide or a biologically active fragmentthereof is contacted with a test compound. In some embodiments, theTHAP-family polypeptide comprises an amino acid- sequence having atleast 30% amino acid identity to an amino acid sequence of SEQ ID NO: 1or SEQ ID NO: 2. Whether the test compound modulates the binding of SLCwith a THAP-family polypeptide, such as THAP1 (SEQ ID NO: 3), isdetermined by determining whether the test compound modulates theactivity of the THAP-family polypeptide or biologically active fragmentthereof. Biologically active framents of a THAP-family polypeptide maybe at least 5, at least 8, at least 10, at least 12, at least 15, atleast i8, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 110, at least 120, at least 130, atleast 140, at least 150, at least 160, at least 170, at least 180, atleast 190, at least 200, at least 210, at least 220 or at least morethan 220 amino acids in length. A determination that the test compoundmodulates the activity of said polypeptide indicates that the testcompound is a candidate modulator of THAP-mediated activities.

Although THAP-family polypeptides, SLC-binding domains of THAP-familypolypeptides, THAP oligomers, and SLC-bindingdomain-THAP1-immunoglobulin fusion proteins can be used for theabove-mentioned SLC interactions, it will be appreciated that homologsof THAP-family polypeptides, SLC-binding domains of THAP-familypolypeptides, THAP oligomers, and SLC-bindingdomain-THAP1-immunoglobulin fusion proteins can be used in place ofTHAP-family polypeptides, SLC-binding domains of THAP-familypolypeptides, THAP oligomers, and SLC-bindingdomain-THAP1-immunoglobulin fusion proteins. For example, homologshaving at least about 30-40% identity, preferably at least about 40-50%identity, more preferably at least about 50-60%, and even morepreferably at least about 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%or 99.8% identity across the amino acid sequences of SEQ ID NOs: 1-114or portions thereof can be used.

Although this section, entitled “Oligomeric Forms of THAP-1,” describesTHAP-family polypeptides, SLC-binding domains of THAP-familypolypeptides, THAP oligomers, SLC-binding domain-THAP1-immunoglobulinfusion proteins and homologs of these polypeptides as well as methods ofusing such polypeptides, it will be appreciated that such polypeptidesare included in the class of THAP-type chemokine-binding agents.Accordingly, the above description also applies to THAP-typechemokine-binding agents. It will be appreciated that THAP-typechemokine-binding agents will be used for applications which include,but are not limited to, chemokine binding, inhibiting or enhancingchemokine activity, chemokine detection, reducing the symptomsassociated with a chemokine influenced or mediated condition, andreducing or preventing inflammation or other chemokine mediatedconditions. THAP-type chemokine-binding agents can also be used in thekits, devices, compositions, and procedures described elsewhere herein.

In some embodiments of the present invention, THAP-typechemokine-binding agents bind to or otherwise modulate the activity ofone or more chemokines selected from the group consisting of XCL1, XCL2,CCL1, CCL2, CCL3, CCL3L1, SCYA3L2, CCL4, CCL4L, CCL5, CCL6, CCL7, CCL8,SCYA9, SCYA10, CCL11, SCYA12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18,CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28,clone 391, CARP CC-1, CCL1, CK-1, regakine-1, K203, CXCL1, CXCL1P,CXCL2, CXCL3, PF4, PF4V1, CXCL5, CXCL6, PPBP, SPBPBP, IL8, CXCL9,CXCL10, CXCL11, CXCL12, CXCL14, CXCL15, CXCL16, NAP-4, LFCA-1, Scyba,JSC, VHSV-induced protein, CX3CL1, and fCL1.

Primers and Probes

Primers and probes of the invention can be prepared by any suitablemethod, including, for example, cloning and restriction of appropriatesequences and direct chemical synthesis by a method such as thephosphodiester method of Narang S A et al (Methods Enzymol1979;68:90-98), the phosphodiester method of Brown E L et al (MethodsEnzymol 1979;68:109-151), the diethylphosphoramidite method of Beaucageet al (Tetrahedron Lett 1981, 22: 1859-1862) and the solid supportmethod described in EP 0 707 592, the disclosures of which areincorporated herein by reference in their entireties.

Detection probes are generally nucleic acid sequences or unchargednucleic acid analogs such as, for example peptide nucleic acids whichare disclosed in International Patent Application WO 92/20702,morpholino analogs which are described in U.S. Pat. Nos. 5,185,444;5,034,506 and 5,142,047. If desired, the probe may be rendered“non-extendable” in that additional dNTPs cannot be added to the probe.In and of themselves analogs usually are non-extendable and nucleic acidprobes can be rendered non-extendable by modifying the 3′ end of theprobe such that the hydroxyl group is no longer capable of participatingin elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group.

Any of the polynucleotides of the present invention can be labeled, ifdesired, by incorporating any label known in the art to be detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include radioactive substances(including, ³²P, ³⁵S, ³H, ¹²⁵I), fluorescent dyes (including,5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) orbiotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends.Examples of non-radioactive labeling of nucleic acid fragments aredescribed in (Urdea et al. (Nucleic Acids Research. 11:4937-4957, 1988)or Sanchez-Pescador et al. (J. Clin. Microbiol. 26(10):1934-1938, 1988).In addition, the probes according to the present invention may havestructural characteristics such that they allow the signalamplification, such structural characteristics being, for example,branched DNA probes as those described by Urdea et al (Nucleic AcidsSymp. Ser. 24:197-200, 1991) or in the European patent No. EP 0 225 807(Chiron).

A label can also be used to capture the primer, so as to facilitate theimmobilization of either the primer or a primer extension product, suchas amplified DNA, on a solid support. A capture label is attached to theprimers or probes and can be a specific binding member which forms abinding pair with the solid's phase reagent's specific binding member(e.g. biotin and streptavidin). Therefore depending upon the type oflabel carried by a polynucleotide or a probe, it may be employed tocapture or to detect the target DNA. Further, it will be understood thatthe polynucleotides, primers or probes provided herein, may, themselves,serve as the capture label. For example, in the case where a solid phasereagent's binding member is a nucleic acid sequence, it may be selectedsuch that it binds a complementary portion of a primer or probe tothereby immobilize the primer or probe to the solid phase. In caseswhere a polynucleotide probe itself serves as the binding member, thoseskilled in the art will recognize that the probe will contain a sequenceor “tail” that is not complementary to the target. In the case where apolynucleotide primer itself serves as the capture label, at least aportion of the primer will be free to hybridize with a nucleic acid on asolid phase. DNA labeling techniques are well known to the skilledtechnician.

The probes of the present invention are useful for a number of purposes.They can be notably used in Southern hybridization to genomic DNA. Theprobes can also be used to detect PCR amplification products. They mayalso be used to detect mismatches in a THAP-family gene or mRNA usingother techniques.

Any of the nucleic acids, polynucleotides, primers and probes of thepresent invention can be conveniently immobilized on a solid support.Solid supports are known to those skilled in the art and include thewalls of wells of a reaction tray, test tubes, polystyrene beads,magnetic beads, nitrocellulose strips, membranes, microparticles such aslatex particles, sheep (or other animal) red blood cells, duracytes andothers. The solid support is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and duracytes are all suitable examples. Suitable methods forimmobilizing nucleic acids on solid phases include ionic, hydrophobic,covalent interactions and the like. A solid support, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid support can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid support and which has the ability to immobilize thecapture reagent through a specific binding reaction. The receptormolecule enables the indirect binding of the capture reagent to a solidsupport material before the performance of the assay or during theperformance of the assay. The solid phase thus can be a plastic,derivatized plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microtiter well, sheet, bead, microparticle,chip, sheep (or other suitable animal's) red blood cells, duracytes andother configurations known to those of ordinary skill in the art. Thenucleic acids, polynucleotides, primers and probes of the invention canbe attached to or immobilized on a solid support individually or ingroups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinctpolynucleotides of the invention to a single solid support. In addition,polynucleotides other than those of the invention may be attached to thesame solid support as one or more polynucleotides of the invention.

Any polynucleotide provided herein may be attached in overlapping areasor at random locations on a solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention. One particular embodiment of thesepolynucleotide arrays is known as the Genechips, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092, the disclosures of which are incorporated herein by referencein their entireties.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a THAP family orTHAP domain polypeptide, or a biologically active fragment or homologuethereof.

Vectors may have particular use in the preparation of a recombinantprotein of the invention, or for use in gene therapy. Gene therapypresents a means to deliver a THAP family or THAP domain polypeptide, ora biologically active fragment or homologue thereof to a subject inorder to regulate apoptosis for treatment of a disorder.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise aTHAP-family or THAP domain nucleic acid of the invention in a formsuitable for expression of the nucleic acid in a host cell, which meansthat the recombinant expression vectors include one or more regulatorysequences, selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid sequence tobe expressed. Within a recombinant expression vector, “operably linked”is intended to mean that the nucleotide sequence of interest is linkedto the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990), the disclosure of which is incorporatedherein by reference in its entirety. Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., THAP-family proteins, mutant forms of THAP-familyproteins, fusion proteins, or fragments of any of the precedingproteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof in prokaryotic oreukaryotic cells. For example, THAP-family or THAP domain proteins canbe expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990), the disclosure of which is incorporated herein by reference inits entirety. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), thedisclosures of which are incorporated herein by reference in theirentireties, which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

Purified fusion proteins can be utilized in THAP-family activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for THAP-family or THAP domainproteins, for example. In a preferred embodiment, a THAP-family or THAPdomain fusion protein expressed in a retroviral expression vector of thepresent invention can be utilized to infect bone marrow cells which aresubsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed(for example, six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET Ild (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89), the disclosures of which areincorporated herein by reference in their entireties. Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase(T7 gn 1). This viral polymerase is supplied by host strains BL21 (DE3)or HMS174(DE3) from a resident prophage harboring a T7 gnl gene underthe transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128, the disclosure of which is incorporatedherein by reference in its entirety). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., (1992) NucleicAcids Res. 20:2111-2118, the disclosure of which is incorporated hereinby reference in its entirety). Such alteration of nucleic acid sequencesof the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the THAP-family expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec 1 (Baldari, et al., (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.), the disclosuresof which are incorporated herein by reference in their entireties.

Alternatively, THAP-family or THAP domain proteins can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39), the disclosures of which are incorporated herein byreference in their entireties. In particularly preferred embodiments,THAP-family proteins are expressed according to Karniski et al, Am. J.Physiol. (1998) 275: F79-87, the disclosure of which is incorporatedherein by reference in its entirety.

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195), thedisclosures of which are incorporated herein by reference in theirentireties. When used in mammalian cells, the expression vector'scontrol functions are often provided by viral regulatory elements. Forexample, commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. For other suitable expressionsystems for both prokaryotic and eukaryotic cells see chapters 16 and 17of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, the disclosureof which is incorporated herein by reference in its entirety. In anotherembodiment, the recombinant mammalian expression vector is capable ofdirecting expression of the nucleic acid preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe nucleic acid). Tissue-specific regulatory elements are known in theart, and are further described below.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to THAP-family mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986, the disclosure of which isincorporated herein by reference in its entirety.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such term refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aTHAP-family protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells or human cells). Other suitable host cells areknown to those skilled in the art, including mouse 3T3 cells as furtherdescribed in the Examples.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, thedisclosure of which is incorporated herein by reference in itsentirety), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a THAP-family protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a THAP-familyprotein. Accordingly, the invention further provides methods forproducing a THAP-family protein using the host cells of the invention.In one embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding aTHAP-family protein has been introduced) in a suitable medium such thata THAP-family protein is produced. In another embodiment, the methodfurther comprises isolating a THAP-family protein from the medium or thehost cell.

In another embodiment, the invention encompassesa method comprising:providing a cell capable of expressing a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof,culturing said cell in a suitable medium such that a THAP-family or THAPdomain protein is produced, and isolating or purifying the THAP-familyor THAP domain protein from the medium or cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals, such as for the study of disorders in which THAPfamily proteins are implicated. For example, in one embodiment, a hostcell of the invention is a fertilized oocyte or an embryonic stem cellinto which THAP-family- or THAP domain- coding sequences have beenintroduced. Such host cells can then be used to create non-humantransgenic animals in which exogenous THAP-family or THAP domainsequences have been introduced into their genome or homologousrecombinant animals in which endogenous THAP-family or THAP domainsequences have been altered. Such animals are useful for studying thefunction and/or activity of a THAP-family or THAP domain polypeptide orfragment thereof and for identifying and/or evaluating modulators of aTHAP-family or THAP domain activity. As used herein, a “transgenicanimal” is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous THAP-family or THAP domaingene has been altered by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986, the disclosures of which are incorporated herein by reference intheir entireties).

Gene Therapy Vectors

Prefered vectors for administration to a subject can be constructedaccording to well known methods. Vectors will comprise regulatoryelements (e.g. promotor, enhancer, etc) capable of directing theexpression of the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, P actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Severalinducible promoter systems are available for production of viral vectorswhere the transgene product may be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constituitively expressedfrom one vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A. Anotherinducible system that would be useful is the Tet-Off or Tet On system(Clontech, Palo Alto, Calif.) originally developed by Gossen and Bujard(Gossen and Bujard, 1992; Gossen et al, 1995). This system also allowshigh levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off system, gene expression is turned onin the absence of doxycycline. These systems are based on two regulatoryelements derived from the tetracycline resistance operon of E. coli. Thetetracycline operator sequence to which the tetracycline repressorbinds, and the tetracycline repressor protein. The gene of interest iscloned into a plasmid behind a promoter that has tetracycline-responsiveelements present in it. A second plasmid contains a regulatory elementcalled the tetracycline-controlled transactivator, which is composed, inthe Tet Off system, of the VP16 domain from the herpes simplex virus andthe wild-type tertracycline repressor.

Thus in the absence of doxycycline, transcription is constituitively on.In the Tet-OnTm system, the tetracycline repressor is not wild-type andin the presence of doxycycline activates transcription. For gene therapyvector production, the Tet Off system would be preferable so that theproducer cells could be grown in the presence of tetracycline ordoxycycline and prevent expression of a potentially toxic transgene, butwhen the vector is introduced to the patient, the gene expression wouldbe constituitively on.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic_cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HfV-2 LTR, adenoviruspromoters such as from the EIA, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcriptionin specific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate. Similarly, promoters as follows may be used to target geneexpression in other tissues.

Tissue specific promoters include in (a) pancreas: insulin, elastin,amylase, pdr-I, pdx-I, glucokinase; (b) liver: albumin PEPCK, HBVenhancer, alpha fetoprotein, apolipoprotein C, alpha-I antitrypsin,vitellogenin, NF-AB, Transthyretin; (c) skeletal muscle: myosin H chain,muscle creatine kinase, dystrophin, calpain p94, skeletal alpha-actin,fast troponin 1; (d) skin: keratin K6, keratin KI; (e) lung: CFTR, humancytokeratin IS (K 18), pulmonary surfactant proteins A, B and C, CC-10,Pi; (f) smooth muscle: sm22 alpha, SM-alpha-actin; (g) endothelium:endothelin-I, E-selectin, von Willebrand factor, TIE (Korhonen et al.,1995), KDR/flk-I; (h) melanocytes: tyrosinase; (i) adipose tissue:lipoprotein lipase (Zechner et al., 1988), adipsin (Spiegelman et al.,1989), acetyl-CoA carboxylase (Pape and Kim, 1989), glycerophosphatedehydrogenase (Dani et al., 1989), adipocyte P2 (Hunt et al., 1986); and(j) blood: P-globin.

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example in gene therapy applications where theindication is in a gonadal tissue where specific steroids are producedor routed to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV,MT-1, ecdysone and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary and adrenal hormones are expectedto be useful in the present invention. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible byphorbol esters, TNF alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-I antichymotrypsin.

It is envisioned that cell cycle regulatable promoters may be useful inthe present invention. For example, in a bi-cistronic gene therapyvector, use of a strong CMV promoter to drive expression of a first genesuch as p16 that arrests cells in the G1 phase could be followed byexpression of a second gene such as p53 under the control of a promoterthat is active in the GI phase of the cell cycle, thus providing a“second hit” that would push the cell into apoptosis. Other promoterssuch as those of various cyclins, PCNA, galectin-3, E2FI, p53 and BRCAIcould be used.

Tumor specific promoters such as osteocalcin, hypoxia-responsive element(HRE), NIAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase alsomay be used to regulate gene expression in tumor cells. Other promotersthat could be used according to the present invention includeLac-regulatable, chemotherapy inducible (e.g. MDR), and heat(hyperthermia) inducible promoters, Radiation-inducible (e.g., EGR (Jokiet al., 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acidpromoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, -actin andalpha-globin. Many other promoters that may be useful are listed inWalther and Stein (1996), the disclosure of which is incorporated hereinby reference.

It is envisioned that any of the above promoters alone or in combinationwith another may be useful according to the present invention dependingon the action desired.

In addition, this list of promoters should not be considered to beexhaustive or limiting, those of skill in the art will know of otherpromoters that may be used in conjunction with the THAP-family and THAPdomain nucleic acids and methods disclosed herein.

Enhancers

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters additional to the tissue specific promoterslisted above, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid encoding a gene of interest in an expression construct (list ofenhancers, and Table 1). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

Suitable enhancers include: Immunoglobulin Heavy Chain; ImmunoglobulinLight Chain; T-Cell Receptor; HLA DQ (x and DQ beta; beta-Interferon;Interleukin-2; Interleukin-2 Receptor; MHC Class II 5; MHC Class IIHLA-DRalpha; beta-Actin; Muscle Creatine Kinase; Prealburnin(Transthyretin); Elastase I; Metallothionein; Collagenase; Albumin Gene;alpha-Fetoprotein; -Globin; beta-Globin; e-fos; c-HA-ras; Insulin;Neural Cell Adhesion Molecule (NCAM); alpha al-Antitrypsin; H2B (TH2B)Histone; Mouse or Type I Collagen; Glucose-Regulated Proteins (GRP94 andGRP78); Rat Growth Hormone; Human Serum Amyloid A (SAA); Troponin I (TN1); Platelet-Derived Growth Factor; Duchenne Muscular Dystrophy; SV40;Polyoma; Retroviruses; THAPilloma Virus; Hepatitis B Virus; HumanImmunodeficiency Virus; Cytomegalovirus; and Gibbon Ape Leukemia Virus.TABLE 1 Element Inducer MT 11 Phorbol Ester (TPA) Heavy metals MMTV(mouse mammary tumor Glucocorticoids virus) B-Interferon poly(rI)X;poly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H2O2 H202Collagenase Phorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1SV40 Phorbol Ester (TPA) Murine MX Gene Interferon, Newcastle DiseaseVirus GRP78 Gene A23187 oc-2-Macroglobulin IL-6 Vimentin Serum NMC ClassI Gene H-2 kB Interferon HSP70 Ela, SV40 Large T Antigen Insulin E BoxGlucose Proliferin Phorbol Ester-TPA Tumor Necrosis Factor FMA ThyroidStimulating Hormone alpha Gene Thyroid Hormone

In preferred embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells (Ridgeway, 1988;Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986,the disclosures of which are incorporated herein by reference). Thefirst viruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). These have a relatively low capacityfor foreign DNA sequences and have a restricted host spectrum.

Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kB of foreign genetic material but can be readily introduced in avariety of cell lines and laboratory animals (Nicolas and Rubenstein,1988; Temin, 1986).

(iii) Polyadenylation Signals

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human or bovine growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

Antisense Constructs

The term “antisense nucleic acid” is intended to refer to theoligonucleotides complementary to the base sequences of DNA and RNA.Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport and/or translation. Targetingdouble-stranded (ds) DNA with oligonucleotide leads to triple-helixformation; targeting RNA will lead to double-helix formation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNAs, maybe employed to inhibit gene transcription or translation or both withina host cell, either in vitro or in vivo, such as within a host animal,including a human subject. Nucleic acid sequences comprisingcomplementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementary rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, micleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only single or double mismatches. Naturally, nucleic acidsequences which are “completely complementary” will be nuleic acidsequences which are entirely complementary throughout their entirelength and have no base mismatches.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence 17 bases long shouldoccur only once in the human genome and, therefore, suffice to specify aunique target sequence.

Although shorter oligomers are easier to make and increase in vivoaccessibility, numerous other factors are involved in determining thespecificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that oligonucleotides of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will beused. One can readily determine whether a given antisense nucleic acidis effective at targeting of the corresponding host cell gene simply bytesting the constructs in vitro to determine whether the endogenousgene's function is affected or whether the expression of related geneshaving complementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines.

Oligonucleotides which contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression (Wagner et al, 1993).

Ribozyme Constructs

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in oncogene DNA andRNA. Ribozymes either can be targeted directly to cells, in the form ofRNA oligo-nucleotides incorporating ribozyme sequences, or introducedinto the cell as an expression construct encoding the desired ribozymalRNA. Ribozymes may be used and applied in much the same way as describedfor antisense nucleic acids.

Methods of Gene Transfer

In order to mediate the effect of transgene expression in a cell, itwill be necessary to transfer the therapeutic expression constructs ofthe present invention into a cell. This section provides a discussion ofmethods and compositions of viral production and viral gene transfer, aswell as non-viral gene transfer methods.

(i) Viral Vector-Mediated Transfer

The THAP-family gene is incorporated into a viral infectious particle tomediate gene transfer to a cell. Additional expression constructsencoding other therapeutic agents as described herein may also betransferred via viral transduction using infectious viral particles, forexample, by transformation with an adenovirus vector of the presentinvention as described herein below. Alternatively, retroviral or bovinepapilloma virus may be employed, both of which permit permanenttransformation of a host cell with a gene(s) of interest. Thus, in oneexample, viral infection of cells is used in order to delivertherapeutically significant genes to a cell. Typically, the virus simplywill be exposed to the appropriate host cell under physiologicconditions, permitting uptake of the virus. Though adenovirus isexemplified, the present methods may be advantageously employed withother viral or non-viral vectors, as discussed below.

Adenovirus

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kB viralgenome is bounded by 100-200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The El region (EIA and EIB) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication.

These proteins are involved in DNA replication, late gene expression,and host cell shut off (Renan, 1990). The products of the late genes (LI, L2, U, L4 and L5), including the majority of the viral capsidproteins, are expressed only after significant processing of a singleprimary transcript issued by the major late promoter (MLP). The MLP(located at 16.8 map units) is particularly efficient during the latephase of infection, and all the mRNAs issued from this promoter possessa 5′ tripartite leader (TL) sequence which makes them preferred mRNAsfor translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.The two goals are, to an extent, coterminous in that elimination ofadenoviral genes serves both ends. By practice of the present invention,it is possible achieve both these goals while retaining the ability tomanipulate the therapeutic constructs with relative case.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsidation is localizedbetween 194 385 bp (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage k DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. El substitution vectors of Ad have demonstrated that a450 bp (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts, 1977). Laterstudies showed that a mutant with a deletion in the EIA (194-358 bp)region of the genome grew poorly even in a cell line that complementedthe early (EIA) function (Hearing and Shenk, 1983). When a compensatingadenoviral DNA (0-353 bp) was recombined into the right end of themutant, the virus was packaged normally. Further mutational analysisidentified a short, repeated, position-dependent element in the left endof the Ad5 genome. One copy of the repeat was found to be sufficient forefficient packaging if present at either end of the genome, but not whenmoved towards the interior of the Ad5 DNA molecule (Hearing et al.,1987).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals arepackaged selectively when compared to the helpers. If the preference isgreat enough, stocks approaching homogeneity should be achieved.

Retrovirus

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins.

The integration results in the retention of the viral gene sequences inthe recipient cell and its descendants. The retroviral genome containsthree genes—gag, pol and env—that code for capsid proteins, polymeraseenzyme, and envelope components, respectively. A sequence found upstreamfrom the gag gene, termed T, functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5′ and 3′ ends of the viral genome. These contain strongpromoter and enhancer sequences and also are required for integration inthe host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and T components is constructed (Mann et al.,1983). When a recombinant plasmid containing a human cDNA, together withthe retroviral LTR and T sequences is introduced into this cell line (bycalcium phosphate precipitation for example), the T sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983, the disclosures ofwhich are incorporated herein by reference). The media containing therecombinant retroviruses is collected, optionally concentrated, and usedfor gene transfer. Retroviral vectors are able to infect a broad varietyof cell types. However, integration and stable expression of many typesof retroviruses require the division of host cells (Paskind et al.,1975).

An approach designed to allow specific targeting of retrovirus vectorsrecently was developed based on the chemical modification of aretrovirus by the chemical addition of galactose residues to the viralenvelope. This modification could permit the specific infection of cellssuch as hepatocytes via asialoglycoprotein receptors, should this bedesired.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

Adeno-Associated Virus

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.Inverted terminal repeats flank the genome. Two genes are present withinthe genome, giving rise to a number of distinct gene products. Thefirst, the cap gene, produces three different virion proteins (VP),designated VP-1, VP 2 and VP-3.

The second, the rep gene, encodes four non-structural proteins (NS). Oneor more of these rep gene products is responsible for transactivatingAAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced.

The splice site, derived from map units 42-46, is the same for eachtranscript. The four non-structural proteins apparently are derived fromthe longer of the transcripts, and three virion proteins all arise fromthe smallest transcript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytornegalovirus, pseudorabies virus and, of course, adenovirus.

The best characterized of the helpers is adenovirus, and many “early”functions for this virus have been shown to assist with AAV replication.Low level expression of AAV rep proteins is believed to hold AAVstructural expression in check, and helper virus infection is thought toremove this block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome (Samulski et al, 1987), or by other methods knownto the skilled artisan, including but not limited to chemical orenzymatic synthesis of the terminal repeats based upon the publishedsequence of AAV. The ordinarily skilled artisan can determine, bywell-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e., stableand site specific integration.

The ordinarily skilled artisan also can determine which minormodifications of the sequence can be tolerated while maintaining theability of the terminal repeats to direct stable, site-specificintegration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo (Carter and Flotte,1996; Chattedee et al., 1995; Ferrari et al., 1996; Fisher et al., 1996;Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994; 1996,Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996; Xiaoet al., 1996, the disclosures of which are incorporated herein byreference in their entireties).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1996; Flotte et al., 1993, the disclosures of which areincorporated herein by reference). Similarly, the prospects fortreatment of muscular dystrophy by AAV-mediated gene delivery of thedystrophin gene to skeletal muscle, of Parkinson's disease by tyrosinehydroxylase gene delivery to the brain, of hemophilia B by Factor IXgene delivery to the liver, and potentially of myocardial infarction byvascular endothelial growth factor gene to the heart, appear promisingsince AAV-mediated transgene expression in these organs has recentlybeen shown to be highly efficient (Fisher et al., 1996; Flotte et al.,1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al.,1996; Ping et al., 1996; and Xiao et al., 1996, the disclosures of whichare incorporated herein by reference in their entireties.).

Other Viral Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) andhepatitus B viruses have also been developed and are useful in thepresent invention. They offer several attractive features for variousmammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden,1986; Coupar et al., 1988; and Horwich et al., 1990, the disclosures ofwhich are incorporated herein by reference in their entireties.).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al., recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was cotransfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

In still further embodiments of the present invention, the nucleic acidsto be delivered are housed within an infective virus that has beenengineered to express a specific binding ligand. The virus particle willthus bind specifically to the cognate receptors of the target cell anddeliver the contents to the cell. A novel approach designed to allowspecific targeting of retrovirus vectors was recently developed based onthe chemical modification of a retrovirus by the chemical addition oflactose residues to the viral envelope. This modification can permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

(ii) Non-Viral Transfer

DNA constructs of the present invention are generally delivered to acell. In certain situations, the nucleic acid to be transferred isnon-infectious, and can be transferred using non-viral methods.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication(Fechheimer et al., 1987), gene bombardment using high velocitymicroprojectiles (Yang et al., 1990), and receptor-mediated transfection(Wu and Wu, 1987; Wu and Wu, 1988), the disclosures of which areincorporated herein by reference in their entireties.

Once the construct has been delivered into the cell the nucleic acidencoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.Thi-s integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

How the expression construct is delivered to a cell and where in thecell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In a particular embodiment of the invention, the expression constructmay be entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). The addition of DNA to cationic liposomes causes atopological transition from liposomes to optically birefringentliquid-crystalline condensed globules (Radler et al., 1997). TheseDNA-lipid complexes are potential non-viral vectors for use in genetherapy.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Using the P-lactamase gene, Wong et al.(1980) demonstrated the feasibility of liposome-mediated delivery andexpression of foreign DNA in cultured chick embryo, HeLa, and hepatomacells. Nicolau et al. (1987) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection. Also included arevarious commercial approaches involving “lipofection” technology.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989).

In other embodiments, the liposome may be complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Katoet al., 1991). In yet further embodiments, the liposome may be complexedor employed in conjunction with both HVJ and HMG-1. In that suchexpression constructs have been successfully employed in transfer andexpression of nucleic acid in vitro and in vivo, then they areapplicable for the present invention.

Other vector delivery systems which can be employed to deliver a nucleicacid encoding a therapeutic gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor mediated endocytosis in almost all eukaryoticcells. Because of the cell type specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferring (Wagner et al., 1990).

Recently, a synthetic neoglycoprotein, which recognizes the samereceptor as ASOR, has been used as a gene delivery vehicle (Ferkol etal., 1993; Perales et al., 1994) and epidermal growth factor (EGF) hasalso been used to deliver genes to squamous carcinoma cells (Myers, EPO0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al, (1987) employed lactosyl-ceramide,a galactose terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a therapeutic genealso may be specifically delivered into a cell type such as prostate,epithelial or tumor cells, by any number of receptor-ligand systems withor without liposomes. For example, the human prostate-specific antigen(Watt et al, 1986) may be used as the receptor for mediated delivery ofa nucleic acid in prostate tissue.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al, (1984) successfullyinjected polyornavirus DNA in the form of CaP04 precipitates into liverand spleen of adult and newborn mice demonstrating active viralreplication and acute infection.

Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaP04 precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga CAM may also be transferred in a similar manner in vivo and expressCAM.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al, 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical cur-rent, which inturn provides the motive force (Yang et al, 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Antibodies Polyclonal anti-THAP-family or anti-THAP domain antibodiescan be prepared as described above by immunizing a suitable subject witha THAP-family or THAP domain immunogen. The anti-THAP-family or anti-THAP domain antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized THAP-family or THAP domainprotein. If desired, the antibody molecules directed against THAP-familycan be isolated from the mammal (e.g., from the blood) and furtherpurified by well known techniques, such as protein A chromatography toobtain the IgG fraction. At an appropriate time after immunization,e.g., when the anti-THAP-family antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thosedescribed in the following references, the disclosures of which areincorporated herein by reference in their entireties: the hybridomatechnique originally described by Kohler and Milstein (1975) Nature256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46;Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) PNAS76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the morerecent human B cell hybridoma technique (Kozbor et al. (1983) ImmunolToday 4:72), the EBV-hybridoma technique (Cole et al. (1985), MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triomatechniques. The technology for producing monoclonal antibody hybridomasis well known (see generally R. H. Kenneth, in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L.Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortalcell line (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with a THAP-family immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds THAP-family.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-THAP-family or anti-THAP domain monoclonal antibody (see, e.g., G.Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic CellGenet., cited supra; Lerner, Yale J Biol. Med, cited supra; Kenneth,Monoclonal Antibodies, cited supra), the disclosures of which areincorporated herein by reference in their entireties. Moreover, theordinarily skilled worker will appreciate that there are many variationsof such methods which also would be useful. Typically, the immortal cellline (e.g., a myeloma cell line) is derived from the same mammalianspecies as the lymphocytes. For example, murine hybridomas can be madeby fusing lymphocytes from a mouse immunized with an immunogenicpreparation of the present invention with an immortalized mouse cellline. Preferred immortal cell lines are mouse myeloma cell lines thatare sensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind a THAP-family or THAP domain protein, e.g.,using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-THAP-family or anti-THAP domain antibody can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withTHAP-family or THAP domain protein to thereby isolate immunoglobulinlibrary members that bind THAP-family or THAP domain proteins. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™. Phage Display Kit,Catalog No. 240612), the disclosures of which are incorporated herein byreference in their entireties. Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No.WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/2079 1 Markland et al.PCT International Publication No. WO 92/15679; Breitling et al. PCTInternational Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137;Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature(1990) 348:552-554, the disclosures of which are incorporated herein byreference in their entireties.

Additionally, recombinant anti-THAP-family or anti-THAP domainantibodies, such as chimeric and humanized monoclonal antibodies,comprising both human and non-human portions, which can be made usingstandard recombinant DNA techniques, are within the scope of theinvention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, M., European Patent Application 171496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al.(1987) J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060, the disclosures ofwhich are incorporated herein by reference in their entireties.

An anti-THAP-family of anti-THAP domain antibody (e.g., monoclonalantibody) can be used to isolate THAP-family or THAP domain protein bystandard techniques, such as affinity chromatography orimmunoprecipitation. For example, an anti-THAP-family antibody canfacilitate the purification of natural THAP-family from cells and ofrecombinantly produced THAP-family expressed in host cells. Moreover, ananti-THAP-family antibody can be used to detect THAP-family protein(e.g., in a cellular lysate or cell supernatant) in order to evaluatethe abundance and pattern of expression of the THAP-family protein.Anti-THAP-family antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, -galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include 125 I, 131 1, 35 Sor 3 H.

Drug Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., preferably small molecules, but also peptides,peptidomimetics or other drugs) which bind to THAP-family or THAP domainproteins, have an inhibitory or activating effect on, for example,THAP-family expression or preferably THAP-family activity, or have aninhibitory or -activating effect on, for example, the activity of anTHAP-family target molecule. In some embodiments small molecules can begenerated using combinatorial chemistry or can be obtained from anatural products library. Assays may be cell based, non-cell-based or invivo assays. Drug screening assays may be binding assays or morepreferentially functional assays, as further described.

In general, any suitable activity of a THAP-family protein can bedetected in a drug screening assay, including: (1) mediating apoptosisor cell proliferation when expressed or introduced into a cell, mostpreferably inducing or enhancing apoptosis, and/or most preferablyreducing cell proliferation; (2) mediating apoptosis or cellproliferation of an endothelial cell; (3) mediating apoptosis or cellproliferation of a hyperproliferative cell; (4) mediating apoptosis orcell proliferation of a CNS cell, preferably a neuronal or glial cell;(5) an activity indicative of a biological function in an animalselected from the group consisting of mediating, preferably inhibitingangiogenesis, mediating, preferably inhibiting inflammation, inhibitionof metastatic potential of cancerous tissue, reduction of tumor burden,increase in sensitivity to chemotherapy or radiotherapy, killing acancer cell, inhibition of the growth of a cancer cell, or induction oftumor regression; or (6) interaction with a THAP family target moleculeor THAP domain target molecule, preferably interaction with a protein ora nucleic acid.

The invention also provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., preferably small molecules, but alsopeptides, peptidomimetics or other drugs) which bind to THAP1, PAR4 orPML-NB proteins, and have an inhibitory or activating effect on PAR4 orTHAP1 recruitment or binding to or association with PML-NBs orinteraction, such as binding, of SLC with a THAP-family polypeptide or acellular response to SLC which is mediated by a THAP-family polypeptide.

In one embodiment, the invention provides assays for screening candidateor test compounds which are target molecules of a THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof. In another embodiment, the invention provides assays forscreening candidate or test compounds which bind to or modulate theactivity of a THAP family or THAP domain polypeptide, or a biologicallyactive fragment or homologue thereof. The test compounds of the presentinvention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis used with peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145, thedisclosure of which is incorporated herein by reference in itsentirety).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233, the disclosures ofwhich are incorporated herein by reference in their entireties.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.), the disclosures of which are incorporated herein by referencein their entireties.

Determining the ability of the test compound to inhibit or increaseTHAP-family polypeptide activity can also be accomplished, for example,by coupling the THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof with a radioisotope orenzymatic label such that binding of the THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof toits cognate target molecule can be determined by detecting the labeledTHAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof in a complex. For example, compounds(e.g., THAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. The labeled molecule is placed incontact with its cognate molecule and the extent of complex formation ismeasured. For example, the extent of complex formation may be measuredby immuno precipitating the complex or by performing gelelectrophoresis.

It is also within the scope of this invention to determine the abilityof a compound (e.g., THAP family or THAP domain polypeptide, orbiologically active fragment or homologue thereof) to interact with itscognate target molecule without the labeling of any of the interactants.For example, a microphysiometer can be used to detect the interaction ofa compound with its cognate target molecule without the labeling ofeither the compound or the target molecule. McConnell, H. M. et al.(1992) Science 257:1906-1912, the disclosure of which is incorporatedherein by reference in its entirety. A microphysiometer such as acytosensor is an analytical instrument that measures the rate at which acell acidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between compound and cognate targetmolecule.

In a preferred embodiment, the assay comprises contacting a cell whichexpresses a THAP family or THAP domain polypeptide, or biologicallyactive fragment or homologue thereof, with a THAP-family or THAP domainprotein target molecule to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to inhibit or increase the activity of the THAP family or THAPdomain polypeptide, or biologically active fragment or homologuethereof, wherein determining the ability of the test compound to inhibitor increase the activity of the THAP family or THAP domain polypeptide,or biologically active fragment or homologue thereof, comprisesdetermining the ability of the test compound to inhibit or increase abiological activity of the THAP-family polypeptide expressing cell.

In another embodiment, the assay comprises contacting a cell whichexpresses a THAP family or THAP domain polypeptide, or biologicallyactive fragment or homologue thereof, with a test compound, anddetermining the ability of the test compound to inhibit or increase theactivity of the THAP family or THAP domain polypeptide, or biologicallyactive fragment or homologue thereof, wherein determining the ability ofthe test compound to inhibit or increase the activity of the THAP familyor THAP domain polypeptide, or biologically active fragment or homologuethereof, comprises determining the ability of the test compound toinhibit or increase a biological activity of the THAP-family polypeptideexpressing cell.

In another preferred embodiment, the assay comprises contacting a cellwhich is responsive to a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof, with a THAP-familyprotein or biologically-active portion thereof, to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to modulate the activity ofthe THAP-family protein or biologically active portion thereof, whereindetermining the ability of the test compound to modulate the activity ofthe THAP-family protein or biologically active portion thereof comprisesdetermining the ability of the test compound to modulate a biologicalactivity of the THAP-family polypeptide-responsive cell (e.g.,determining the ability of the test compound to modulate a THAP-familypolypeptide activity.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a THAP-family target molecule (i.e. amolecule with which THAP-family polypeptide interacts) with a testcompound and determining the ability of the test compound to modulate(e.g. stimulate or inhibit) the activity of the THAP-family targetmolecule. Determining the ability of the test compound to modulate theactivity of a THAP-family target molecule can be accomplished, forexample, by determining the ability of the THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof tobind to or interact with the THAP-family target molecule.

Determining the ability of the THAP family or THAP domain polypeptide,or a biologically active fragment or homologue thereof to bind to orinteract with a THAP-family target molecule can be accomplished by oneof the methods described above for determining direct binding. In apreferred embodiment, determining the ability of the THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof to bind to or interact with a THAP-family target molecule can beaccomplished by determining the activity of the target molecule. Forexample, the activity of the target molecule can be determined bycontacting the target molecule with the THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof andmeasuring induction of a cellular second messenger of the target (i.e.intracellular Ca²+, diacylglycerol, IP₃, etc.), detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response, for example, signal transduction orprotein:protein interactions.

In yet another embodiment, an assay of the present invention is acell-free assay in which a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof is contacted with atest compound and the ability of the test compound to bind to the THAPfamily or THAP domain polypeptide, or a biologically active fragment orhomologue thereof is determined. Binding of the test compound to theTHAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof with a known compoundwhich binds THAP-family polypeptide (e.g., a THAP-family targetmolecule) to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof, wherein determiningthe ability of the test compound to interact with a THAP-family proteincomprises determining the ability of the test compound to preferentiallybind to THAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a THAPfamily or THAP domain polypeptide, or a biologically active fragment orhomologue thereof is contacted with a test compound and the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activityof the THAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof is determined. Determining the ability ofthe test compound to modulate the activity of a THAP-family protein canbe accomplished, for example, by determining the ability of the THAPfamily or THAP domain polypeptide, or a biologically active fragment orhomologue thereof to bind to a THAP-family target molecule by one of themethods described above for determining direct binding. Determining theability of the THAP family or THAP domain polypeptide, or a biologicallyactive fragment or homologue thereof to bind to a THAP-family targetmolecule can also be accomplished using a technology such as real-timeBiomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky,C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.Struct. Biol. 5:699-705, the disclosures of which are incorporatedherein by reference in their entireties. As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof canbe accomplished by determining the ability of the THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof to further modulate the activity of a downstream effector (e.g.,a growth factor mediated signal transduction pathway component) of aTHAP-family target molecule. For example, the activity of the effectormolecule on an appropriate target can be determined or the binding ofthe effector to an appropriate target can be determined as previouslydescribed.

In yet another embodiment, the cell-free assay involves contacting aTHAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof with a known compound which binds theTHAP-family protein to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with the THAP-family protein, wherein determiningthe ability of the test compound to interact with the THAP-familyprotein comprises determining the ability of the THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof to preferentially bind to or modulate the activity of aTHAP-family target molecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins (e.g. THAPfamily or THAP domain polypeptide, or a biologically active fragment orhomologue thereof or molecules to which THAP-family targets bind). Inthe case of cell-free assays in which a membrane-bound form an isolatedprotein is used it may be desirable to utilize a solubilizing agent suchthat the membrane-bound form of the isolated protein is maintained insolution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton.[RTM. X-100, Triton.RTM. X-114,Thesit.RTM.], Isotridecypoly(ethylene glycolether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate(CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propanesulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propanesulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof or a target molecule thereof to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound toa THAP family or THAP domain polypeptide, or a biologically activefragment or homologue thereof, or interaction of a THAP-family proteinwith a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example,glutathione-S-transferase/THAP-family fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or THAP-family protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofTHAP-family polypeptide binding or activity determined using standardtechniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either-aTHAP-family protein or a THAP-family target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. BiotinylatedTHAP-family protein or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with a THAP-family protein or targetmolecule but which do not interfere with binding of the THAP-familyprotein to its target molecule can be derivatized to the wells of theplate, and unbound target or THAP-family protein trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with theTHAP-family protein or target molecule, as well as enzyme-linked assayswhich rely on detecting an enzymatic activity associated with theTHAP-family protein or target molecule.

In another embodiment, modulators of THAP-family or THAP domainpolypeptides expression are identified in a method wherein a cell iscontacted with a candidate compound and the expression of THAP-family orTHAP domain polypeptides mRNA or protein in the cell is determined. Thelevel of expression of THAP-family polypeptide mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of THAP-family polypeptide or THAP domain mRNA or protein inthe absence of the candidate compound. The candidate compound can thenbe identified as a modulator of THAP-family polypeptide expression basedon this comparison. For example, when expression of THAP-familypolypeptide or THAP domain mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofTHAP-family polypeptide or THAP domain mRNA or protein expression.Alternatively, when expression of THAP-family polypeptide or THAP domainmRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of THAP-family polypeptide orTHAP domain mRNA or protein expression. The level of THAP-familypolypeptide or THAP domain mRNA or protein expression in the cells canbe determined by methods described herein for detecting THAP-familypolypeptide or THAP domain mRNA or protein.

In yet another aspect of the invention, the THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assayusing the methods described above for use in THAP-familypolypeptide/PAR4 interactions assays, to identify other proteins whichbind to or interact with THAP-family polypeptide (“THAP-family-bindingproteins” or “THAP-family-bp”) and are involved in THAP-familypolypeptide activity. Such THAP-family- or THAP domain-binding proteinsare also likely to be involved in the propagation of signals by theTHAP-family or THAP domain proteins or THAP-family or THAP domainproteins targets as, for example, downstream elements of a THAP-familypolypeptide- or THAP domain-mediated signaling pathway. Alternatively,such THAP-family-binding proteins are likely to be THAP-familypolypeptides inhibitors.

THAP/DNA BINDING ASSAYS

In another embodiment of the invention a method is provided foridentifying compounds which interfere with THAP-family DNA bindingactivity, comprising the steps of: contacting a THAP-family protein or aportion thereof immobilized on a solid support with both a test compoundand DNA fragments, or contacting a DNA fragment immobilized on a solidsupport with both a test compound and a THAP-family protein. The bindingbetween DNA and the THAP-protein or a portion thereof is detected,wherein a decrease in DNA binding when compared to DNA binding in theabsence of the test compound indicates that the test compound is aninhibitor of THAP-family DNA binding activity, and an increase in DNAbinding when compared to DNA binding in the absence of the test compoundindicates that the test compound is an inducer of or restoresTHAP-family DNA binding activity. As discussed further, DNA fragmentsmay be selected to be specific THAP-family protein target DNA obtainedfor example as described in Example 28, or may be non-specificTHAP-family target DNA. Methods for detecting protein-DNA interactiosare well known in the art, including most commonly used electrophoreticmobility shift assays (EMSAs) or by filter binding (Zabel et al, (1991)J. Biol. Chem., 266:252; and Okamoto and Beach, (1994) Embo J. 13:4816). Other assays are available which are amenable for high throughputdetection and quantification of specific and nonspecific DNA binding(Amersham, N.J.; and Gal S. et al, 6^(th) Ann. Conf. Soc. Biomol.Screening, 6-9 Sep. 2000, Vancouver, B.C.).

In a first aspect, a screening assay involves identifying compoundswhich interfere with THAP-family DNA binding activity without priorknowledge about specific THAP-family binding sequences. For example, aTHAP-family protein is contacted with both a test compound and a libraryof oligonucleotides or a sample of DNA fragments not selected based onspecific DNA sequences. Preferably the THAP-family protein isimmobilized on a solid support (such as an array or a column). UnboundDNA is separated from DNA which is bound to the THAP-famliy protein, andthe DNA which is bound to THAP-family protein is detected and can bequantitated by any means known in the art. For example, the DNA fragmentis labelled with a detectable moiety, such as a radioactive moiety, acolorimetric moiety or a fluorescent moiety. Techniques for so labellingDNA are well known in the art.

The DNA which is bound to the THAP-family protein or a portion thereofis separated from unbound DNA by immunoprecipitation with antibodieswhich are specific for the THAP-family protein or a portion thereof. Useof two different monoclonal anti-THAP-family antibodies may result inmore complete immunoprecipitation than either one alone. The amount ofDNA which is in the immunoprecipitate can be quantitated by any meansknown in the art. THAP-family proteins or portions thereof which bind tothe DNA can also be detected by gel shift assays (Tan, Cell, 62:367,1990), nuclease protection assays, or methylase interference assays.

It is still another object of the invention to provide methods foridentifying compounds which restore the ability of mutant THAP-familyproteins or portions thereof to bind to DNA sequences. In one embodimenta method of screening agents for use in therapy is provided comprising:measuring the amount of binding of a THAP-family protein or a portionthereof which is encoded by a mutant gene found in cells of a patient toDNA molecules, preferably random oligonucleotides or DNA fragments froma nucleic acid library; measuring the amount of binding of saidTHAP-family protein or a portion thereof to said nucleic acid moleculesin the presence of a test substance; and comparing the amount of bindingof the THAP-family protein or a portion thereof in the presence of saidtest substance to the amount of binding of the THAP-family protein inthe absence of said test substance, a test substance which increases theamount of binding being a candidate for use in therapy.

In another embodiment of the invention, oligonucleotides can be isolatedwhich restore to mutant THAP-family proteins or portions thereof theability to bind to a consensus binding sequence or conforming sequences.Mutant THAP-family protein or a portion thereof and randomoligonucleotides are added to a solid support on whichTHAP-family-specific DNA fragments are immobilized. Oligonucleotideswhich bind to the solid support are recovered and analyzed. Those whosebinding to the solid support is dependent on the presence of the mutantTHAP-family protein are presumptively binding the support by binding toand restoring the conformation of the mutant protein.

If desired, specific binding can be distinguished from non-specificbinding by any means known in the art. For example, specific bindinginteractions are stronger than non-specific binding interactions. Thusthe incubation mixture can be subjected to any agent or condition whichdestabilizes protein/DNA interactions such that the specific bindingreaction is the predominant one detected. Alternatively, as taught morespecifically below, a non-specific competitor, such as dI-dC, can beadded to the incubation mixture. If the DNA containing the specificbinding sites is labelled and the competitor is unlabeled, then thespecific binding reactions will be the ones predominantly detected uponmeasuring labelled DNA.

According to another embodiment of the invention, after incubation ofTHAP-family protein or a portion thereof with specific DNA fragments allcomponents of the cell lysate which do not bind to the DNA fragments areremoved. This can be accomplished, among other ways, by employing DNAfragments which are attached to an insoluble polymeric support such asagarose, cellulose and the like. After binding, all non-bindingcomponents can be washed away, leaving THAP-family protein or a portionthereof bound to the DNA/solid support. The THAP-family protein or aportion thereof can be quantitated by any means known in the art. It canbe determined using an immunological assay, such as an ELISA, RIA orWestern blotting.

In another embodiment of the invention a method is provided foridentifying compounds which specifically bind toTHAP-family-specific-DNA sequences, comprising the steps of: contactinga THAP-family-specific DNA fragment immobilized on a solid support withboth a test compound and wild-type THAP-family protein or a portionthereof to bind the wild-type THAP-family protein or a portion thereofto the DNA fragment; determining the amount of wild-type THAP-familyprotein which is bound to the DNA fragment, inhibition of binding ofwild-type THAP-family protein by the test compound with respect to acontrol lacking the test compound suggesting binding of the testcompound to the THAP-family-specific DNA binding sequences.

It is still another object of the invention to provide methods foridentifying compounds which restore the ability of mutant THAP-familyproteins or portions thereof to bind to specific DNA binding sequences.In one embodiment a method of screening agents for use in therapy isprovided comprising: measuring the amount of binding of a THAP-familyprotein or a portion thereof which is encoded by a mutant gene found incells of a patient to a DNA molecule which comprises more than onemonomer of a specific THAP-family target nucleotide sequence; measuringthe amount of binding of said THAP-family protein to said nucleic acidmolecule in the presence of a test substance; and comparing the amountof binding of the THAP-family protein in the presence of said testsubstance to the amount of binding of the THAP-family protein or aportion thereof in the absence of said test substance, a test substancewhich increases the amount of binding being a candidate for use intherapy.

In another embodiment of the invention a method is provided forscreening agents for use in therapy comprising: contacting a transfectedcell with a test substance, said transfected cell containing aTHAP-family protein or a portion thereof which is encoded by a mutantgene found in cells of a patient and a reporter gene constructcomprising a reporter gene which encodes an assayable product and asequence which conforms to a THAP-family DNA binding site, wherein saidsequence is upstream from and adjacent to said reporter gene; anddetermining whether the amount of expression of said reporter gene isaltered by the test substance, a test substance which alters the amountof expression of said reporter gene being a candidate for use intherapy.

In still another embodiment a method of screening agents for use intherapy is provided comprising: adding RNA polymerase ribonucleotidesand a THAP-family protein or a portion thereof to a transcriptionconstruct, said transcription construct comprising a reporter gene whichencodes an assayable product and a sequence which conforms to aTHAP-family consensus binding site, said sequence being upstream fromand adjacent to said reporter gene, said step of adding being effectedin the presence and absence of a test substance; determining whether theamount of transcription of said reporter gene is altered by the presenceof said test substance, a test substance which alters the amount oftranscription of said reporter gene being a candidate for use intherapy.

According to the present invention compounds which have THAP-familyactivity are those which specifically complex with aTHAP-family-specific DNA binding site. Oligonucleotides andoligonucleotide containing nucleotide analogs are also contemplatedamong those compounds which are able to complex with aTHAP-family-specific DNA binding site.

Further Assays to Modulate THAP-Family Polypeptide Activity in vivo

It will be appreciated that any suitable assay that allows detection ofTHAP-family polypeptide or THAP domain activity can be used. Examples ofassays for testing protein interaction, nucleic acid binding ormodulation of apoptosis in the presence or absence of a test compoundare further described herein. Thus, the invention encompasses a methodof identifying a candidate THAP-family polypeptide modulator (e.g.activator or inhibitor), said method comprising:

a) providing a cell comprising a THAP family or THAP domain polypeptide,or a biologically active fragment or homolog thereof;

b) contacting said cell with a test compound; and

c) determining whether said compound selectively modulates (e.g.activates or inhibits) THAP-family polypeptide activity, preferablypro-apoptotic activity, or THAP family or THAP domain target binding;wherein a determination that said compound selectively modulates (e.g.activates or inhibits) the activity of said polypeptide indicates thatsaid compound is a candidate modulator (e.g. activator or inhibitorrespectively) of said polypeptide. Preferably, the THAP family or THAPdomain target is a protein or nucleic acid.

Preferably the cell is a cell which has been transfected with anrecombinant expression vector encoding a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof.

Several examples of assays for the detection of apoptosis are describedherein, in the section titled “Apoptosis assays”. Several examples ofassays for the detection of THAP family or THAP domain targetinteractions are described herein, including assays for detection ofprotein interactions and nucleic acid binding.

In one example of an assay for apoptosis activity, a high throughputscreening assay for molecules that abrogate or stimulate THAP-familypolypeptide proapoptotic activity is provided based on serum-withdrawalinduced apoptosis in a 3T3 cell line with tetracycline-regulatedexpression of a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof. Apoptotic cells canbe detected by TUNEL labeling in 96- or 384-wells microplates. A drugscreening assay can be carried out along the lines as described inExample 23. 3T3 cells, which have previously been used to analyze thepro-apoptotic activity of PAR4 (Diaz-Meco et al, 1996; Berra et al.,1997), can be transfected with expression vectors encoding a THAP-familyor THAP domain polypeptide allowing the ectopic expression ofTHAP-family polypeptide. Then, the apoptotic response to serumwithdrawal is assayed in the presence of a test compound, allowing theidentification of test compounds that either enhance or inhibit theability of THAP-family or THAP domain polypeptide to induce apoptosis.Transfected cells are deprived of serum and cells with apoptotic nucleiare counted. Apoptotic nuclei can be counted by DAPI staining and insitu TUNEL assays.

Further THAP-Family Polypeptide/THAP-Target Interaction Assays

In exemplary methods THAP/THAP target interaction assays are describedin the context of THAP1 and the THAP target Par4. However, it will beappreciated that assays for screening for modulators of other THAPfamily members or THAP domains and other THAP target molecules may becarried out by substituting these for THAP1 and Par4 in the methodsbelow. For example, in some embodiments, modulators which affect theinteraction between a THAP-family polypeptide and SLC are identified.

As demonstrated in Examples 4, 5, 6, and 7 and FIGS. 3, 4 and 5, theinventors have demonstrated using several experimental methods thatTHAP1 interacts with the pro-apoptotic protein Par4. In particular, ithas been shown that THAP1 interacts with Par4 wild type (Par4) and aPar4 death domain (Par4DD) in a yeast two-hybrid system. Yeast cellswere cotransformed with BD7-THAP1 and AD7-Par4, AD7, AD7-Par4DD orAD7-Par4Δ expression vectors. Transformants were selected on medialacking histidine and adenine. Identical results were obtained bycotransformation of AD7-THAP1 with BD7-Par4, BD7, BD7-Par4DD orBD7-Par4Δ.

The inventors have also demonstrated in vitro binding of THAP1 toGST-Par4DD. Par4DD was expressed as a GST fusion protein, purified onglutathione sepharose and employed as an affinity matrix for binding ofin vitro translated ³⁵S-methionine labeled THAP1. GST served as negativecontrol.

Futhermore, the inventors have shown that THAP1 interacts with bothPar4DD and SLC in vivo. Myc-Par4DD and GFP-THAP1 expression vectors werecotransfected in primary human endothelial cells. Myc-Par4DD was stainedwith monoclonal anti-myc antibody. Green fluorescence, GFP-THAP1; redfluorescence, Par4DD.

The invention thus encompasses assays for the identification ofmolecules that modulate (stimulate or inhibit) THAP-familypolypeptide/PAR4 binding. In preferred embodiments, the inventionincludes assays for the identification of molecules that modulate(stimulate or inhibit) THAP1 /PAR4 binding or THAP1/SLC binding.

Four examples of high throughput screening assays include:

1) a two hybrid-based assay in yeast to find drugs that disruptinteraction of the THAP-family bait with the PAR4 or SLC as prey

2) an in vitro interaction assay using recombinant THAP-familypolypeptide and PAR4 or SLC proteins

3) a chip-based binding assay using recombinant THAP-family polypeptideand PAR4 or SLC proteins

2) a fluorescence resonance energy transfer (FRET) cell-based assayusing THAP-family polypeptide and PAR4 or SLC proteins fused withfluorescent proteins

The invention thus encompasses a method of identifying a candidateTHAP-family polypeptide/PAR4 or SLC interaction modulator, said methodcomprising:

a) providing a THAP family or THAP domain polypeptide, or a biologicallyactive fragment or homologue thereof and a PAR4 or SLC polypeptide orfragment thereof;

b) contacting said THAP family or THAP domain polypeptide with a testcompound; and

c) determining whether said compound selectively modulates (e.g.activates or inhibits) THAP-family/PAR4 or SLC interaction activity.

Also envisioned is a method comprising:

a) providing a cell comprising a THAP family or THAP domain polypeptide,or a biologically active fragment or homologue thereof and a PAR4 or SLCpolypeptide or fragment thereof;

b) contacting said cell with a test compound; and

c) determining whether said compound selectively modulates (e.g.activates or inhibits) THAP-family/PAR4 or SLC interaction activity.

In general, any suitable assay for the detection of protein-proteininteraction may be used.

In one example, a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof can be used as a “baitprotein” and a PAR4 or SLC protein can be used as a “prey protein” (orvice-versa) in a two-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol.Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300, thedisclosures of which are incorporated herein by reference in theirentireties). The two-hybrid system is based on the modular nature ofmost transcription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a THAP family orTHAP domain polypeptide, or a biologically active fragment or homologuethereof-is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, the genethat codes for a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a THAP-family polypeptide/PAR4 complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with theTHAP-family protein. This assay can thus be carried out in the presenceor absence of a test compound, whereby modulation of THAP-familypolypeptide/PAR4 or SLC interaction can be detected by lower or lack oftranscription of the reported gene.

In other examples, in vitro THAP-family polypeptide/PAR4 or SLCinteraction assays can be carried out, several examples of which arefurther described herein. For example, a recombinant THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof is contacted with a recombinant PAR4 or SLC protein orbiologically active portion thereof, and the ability of the PAR4 or SLCprotein to bind to the THAP-family protein is determined. Binding of thePAR4 or SLC protein compound to the THAP-family protein can bedetermined either directly or indirectly as described herein. In apreferred embodiment, the assay includes contacting the THAP family orTHAP domain polypeptide, or a biologically active fragment or homologuethereof with a PAR4 or SLC protein which binds a THAP-family protein(e.g., a THAP-family target molecule) to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a THAP-family protein,wherein determining the ability of the test compound to interact with aTHAP-family protein comprises determining the ability of the testcompound to preferentially bind to THAP-family or biologically activeportion thereof as compared to the PAR4 or SLC protein. For example, thestep of determining the ability of the test compound to interact with aTHAP-family protein may comprise determining the ability of the compoundto displace Par4 or SLC from a THAP-family protein/Par4 or SLC complexthereby forming a THAP-family protein/compound complex. Alternatively,it will be appreciated that it is also possible to determine the abilityof the test compound to interact with a PAR4 or SLC protein, whereindetermining the ability of the test compound to interact with a PAR4 orSLC protein comprises determining the ability of the test compound topreferentially bind to PAR4 or SLC or biologically active portionthereof as compared to the THAP-family protein. For example, the step ofdetermining the ability of the test compound to interact with aTHAP-family protein may comprise determining the ability of the compoundto displace Par4 or SLC from a THAP-family protein/Par4 or SLC complexthereby forming a THAP-family protein/compound complex.

Assays to Modulate THAP-Family Polypeptide and/or Par4 Trafficking inthe PML Nuclear Bodies (PML NBs)

As demonstrated in Examples 8 and 9, the inventors have demonstratedusing several experimental methods that THAP1 and Par4 localize in PMLNBs.

The inventors demonstrated that THAP1 is a novel protein associated withPML-nuclear bodies. Double immunofluorescence staining showedcolocalization of THAP1 with PML-NBs proteins, PML and Daxx. Primaryhuman endothelial cells were transfected with GFP-THAP1 expressionvector; endogenous PML and Daxx were stained with monoclonal anti-PMLand polyclonal anti-Daxx antibodies, respectively. The inventors alsodemonstrated that Par4 is a novel component of PML-NBs that colocalizeswith THAP1 in vivo by several experiments. In one experiments, doubleimmunofluorescence staining revealed colocalization of Par4 and PML atPML-NBs in primary human endothelial cells or fibroblasts. EndogenousPAR4 and PML were stained with polyclonal anti-PAR4 and monoclonalanti-PML antibodies, respectively. In another experiment, doublestaining revealed colocalization of Par4 and THAP1 in cells expressingectopic GFP-THAP1. Primary human endothelial cells or fibroblasts weretransfected with GFP-THAP1 expression vector; endogenous Par4 wasstained with polyclonal anti-PAR4 antibodies.

The inventors further demonstrated that PML recruits the THAP1/Par4complex to PML-NBs. Triple immunofluorescence staining showedcolocalization of THAP1, Par4 and PML in cells overexpressing PML andabsence of colocalization in cells expressing ectopic Sp100. Hela cellswere cotransfected with GFP-THAP1 and HA-PML or HA-SP100 expressionvectors; HA-PML or HA-S P100 and endogenous Par4 were stained withmonoclonal anti-HA and polyclonal anti-Par4 antibodies, respectively.

Assays to Modulate THAP Family Protein Trafficking in the PML NuclearBodies

Provided are assays for the identification of drugs that modulate(stimulate or inhibit) THAP-family or THAP domain protein, particularlyTHAP1, binding to PML-NB proteins or localization to PML-NBs. Ingeneral, any suitable assay for the detection of protein-proteininteraction may be used. Two examples of high throughput screeningassays include 1) a two hybrid-based assay in yeast to find compoundsthat disrupt interaction of the THAP1 bait with the PML-NB protein prey;and 2) in vitro interaction assays using recombinant THAP1 and PML-NBproteins. Such assays may be conducted as described above with respectto THAP-family/Par4 assays except that the PML-NB protein is used inplace of Par4. Binding may be detected, for example, between aTHAP-family protein and a PML protein or PML associated protein such asdaxx, sp100, sp140, p53, pRB, CBP, BLM or SUMO-1.

Other assays for which standard methods are well known include assays toidentify molecules that modulate, generally inhibit, the colocalizationof THAP1 with PML-NBs. Detection can be carried out using a suitablelabel, such as an anti-THAP1 antibody, and an antibody allowing thedetection of PML-NB protein.

Assays to Modulate PAR4 Trafficking in the PML Bodies

Provided are assays for the identification of drugs that modulate(stimulate or inhibit) PAR4 binding to PML-NB proteins or localizationto PML-NBs. In general, any suitable assay for the detection ofprotein-protein interaction may be used. Two examples of high throughputscreening assays include 1) a two hybrid-based assay in yeast to findcompounds that disrupt interaction of the PAR4 bait with the PML-NBprotein prey; and 2) in vitro interaction assays using recombinant PAR4and PML-NB proteins. Such assays may be conducted as described abovewith respect to THAP-family polypeptide/Par4 assays except that thePML-NB protein is used in place of the THAP-family polypeptide. Bindingmay be detected, for example, between a Par4 protein and a PML proteinor PML associated protein such as daxx, sp100, sp140, p53, pRB, CBP, BLMor SUMO-1.

Other assays for which standard methods are well known include assays toidentify molecules that modulate, generally inhibit, the colocalizationof PAR4 with PML-NBs. Detection can be carried out using a suitablelabel, such as an anti-PAR4 antibody, and an antibody allowing thedetection of PML-NB protein.

This invention further pertains to novel agents identified by theabove-described screening assays and to processes for producing suchagents by use of these assays. Accordingly, in one embodiment, thepresent invention includes a compound or agent obtainable by a methodcomprising the steps of any one of the aforementioned screening assays(e.g., cell-based assays or cell-free assays). For example, in oneembodiment, the invention includes a compound or agent obtainable by amethod comprising contacting a cell which expresses a THAP-family targetmolecule with a test compound and determining the ability of the testcompound to bind to, or modulate the activity of, the THAP-family targetmolecule. In another embodiment, the invention includes a compound oragent obtainable by a method comprising contacting a cell whichexpresses a THAP-family target molecule with a THAP-family protein orbiologically-active portion thereof, to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with, or modulate the activityof, the THAP-family target molecule. In another embodiment, theinvention includes a compound or agent obtainable by a method comprisingcontacting a THAP-family protein or biologically active portion thereofwith a test compound and determining the ability of the test compound tobind to, or modulate (e.g., stimulate or inhibit) the activity of, theTHAP-family protein or biologically active portion thereof. In yetanother embodiment, the present invention includes a compound or agentobtainable by a method comprising contacting a THAP-family protein orbiologically active portion thereof with a known compound which bindsthe THAP-family protein to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with, or modulate the activity of the THAP-familyprotein.

Accordingly, it is within the scope of this invention to further use anagent identified as described herein in an appropriate animal model. Forexample, an agent identified as described herein (e.g., a THAP-family orTHAP domain modulating agent, an antisense THAP-family or THAP domainnucleic acid molecule, a THAP-family- or THAP domain- specific antibody,or a THAP-family- or THAP domain-binding partner) can be used in ananimal model to determine the efficacy, toxicity, or side effects oftreatment with such an agent. Alternatively, an agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

The present invention also pertains to uses of novel agents identifiedby the above-described screening assays for diagnoses, prognoses, andtreatments as described herein. Accordingly, it is within the scope ofthe present invention to use such agents in the design, formulation,synthesis, manufacture, and/or production of a drug or pharmaceuticalcomposition for use in diagnosis, prognosis, or treatment, as describedherein. For example, in one embodiment, the present invention includes amethod of synthesizing or producing a drug or pharmaceutical compositionby reference to the structure and/or properties of a compound obtainableby one of the above-described screening assays. For example, a drug orpharmaceutical composition can be synthesized based on the structureand/or properties of a compound obtained by a method in which a cellwhich expresses a THAP-family target molecule is contacted with a testcompound and the ability of the test compound to bind to, or modulatethe activity of, the THAP-family target molecule is determined. Inanother exemplary embodiment, the present invention includes a method ofsynthesizing or producing a drug or pharmaceutical composition based onthe structure and/or properties of a compound obtainable by a method inwhich a THAP-family protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to, or modulate (e.g., stimulate or inhibit) the activity of, theTHAP-family protein or biologically active portion thereof isdetermined.

Apoptosis Assays

It will be appreciated that any suitable apoptosis assay may be used toassess the apoptotic activity of a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof.

Apoptosis can be recognized by a characteristic pattern ofmorphological, biochemical, and molecular changes. Cells going throughapoptosis appear shrunken, and rounded; they also can be observed tobecome detached from culture dish. The morphological changes involve acharacteristic pattern of condensation of chromatin and cytoplasm whichcan be readily identified by microscopy. When stained with a DNA-bindingdye, e.g., H33258, apoptotic cells display classic condensed andpunctate nuclei instead of homogeneous and round nuclei.

A hallmark of apoptosis is endonucleolysis, a molecular change in whichnuclear DNA is initially degraded at the linker sections of nucleosomesto give rise to fragments equivalent to single and multiple nucleosomes.When these DNA fragments are subjected to gel electrophoresis, theyreveal a series of DNA bands which are positioned approximately equallydistant from each other on the gel. The size difference between the twobands next to each other is about the length of one nucleosome, i.e.,120 base pairs. This characteristic display of the DNA bands is called aDNA ladder and it indicates apoptosis of the cell. Apoptotic cells canbe identified by flow cytometric methods based on measurement ofcellular DNA content, increased sensitivity of DNA to denaturation, oraltered light scattering properties. These methods are well known in theart and are within the contemplation of the invention.

Abnormal DNA breaks which are characteristic of apoptosis can bedetected by any means known in the art. In one preferred embodiment, DNAbreaks are labeled with biotinylated dUTP (b-dUTP). As described in U.S.Pat. No. 5,897,999, the disclosure of which is incorporated herein byreference, cells are fixed and incubated in the presence of biotinylateddUTP with either exogenous terminal transferase (terminal DNAtransferase assay; TdT assay) or DNA polymerase (nick translation assay;NT assay). The biotinylated dUTP is incorporated into the chromosome atthe places where abnormal DNA breaks are repaired, and are detected withfluorescein conjugated to avidin under fluorescence microscopy.

Assessing THAP-Family, THAP Domain and PAR4 Polypeptides Activity

For assessing the nucleic acids and polypeptides of the invention, theapoptosis indicator which is assessed in the screening method of theinvention may be substantially any indicator of the viability of thecell. By way of example, the viability indicator may be selected fromthe group consisting of cell number, cell refractility, cell fragility,cell size, number of cellular vacuoles, a stain which distinguishes livecells from dead cells, methylene blue staining, bud size, bud location,nuclear morphology, and nuclear staining. Other viability indicators andcombinations of the viability indicators described herein are known inthe art and may be used in the screening method of the invention.

Cell death status can be evaluated based on DNA integrity. Assays forthis determination include assaying DNA on an agarose gel to identifyDNA breaking into oligonucleosome ladders and immunohistochemicallydetecting the nicked ends of DNA by labeling the free DNA end withfluorescein or horseradish peroxidase-conjugated UTP via terminaltransferase. Routinely, one can also examine nuclear morphology bypropidium iodide (PI) staining. All three assays (DNA ladder,end-labeling, and PI labelling) are gross measurements and good forthose cells that are already dead or at the end stage of dying.

In a preferred example, an apoptosis assay is based on serum-withdrawalinduced apoptosis in a 3T3 cell line with tetracycline-regulatedexpression of a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof. Detection ofapoptotic cells is accomplished by TUNEL labeling cells in 96- or384-well microplates. This example is further described in Example 23.

In other aspects, assays may test for the generation of cytotoxic deathsignals, anti-viral responses (Tartaglia et al., (1993) Cell74(5):845-531), and/or the activation of acid sphingomyelinase (Wiegmannet al., (1994) Cell 78(6):1005-15) when the THAP-family protein isoverexpressed or ectopically expressed in cells. Assaying for modulationof apoptosis can also be carried out in neuronal cells and lymphocytesfor example, where factor withdrawal is known to induce cell suicide asdemonstrated with neuronal cells requiring nerve growth factor tosurvive (Martin, D. P. et al, (1988) J. Cell Biol 106, 829-844) andlymphocytes depending on a specific lymphokine to live (Kyprianou, N.and Isaacs, J. T. (1988) Endrocrinology 122:552-562). The abovedisclosures are incorporated herein by reference.

THAP-Family or THAP Domain Polypeptide-Marker Fusions in Cell Assays

In one method, an expression vector encoding the a THAP family or THAPdomain polypeptide, or a biologically active fragment or homologuethereof can be used to evaluate the ability of the polypeptides of theinvention to induce apoptosis in cells. If desired, a THAP-family orTHAP domain polypeptide may be fused to a detectable marker in order tofacilitate identification of those cells expressing the a THAP family orTHAP domain polypeptide, or a biologically active fragment or homologuethereof. For example, a variant of the Aequoria victoria GFP variant,enhanced green fluorescent protein (EGFP), can be used in fusion proteinproduction (CLONTECH Laboratories, Inc., 1020 East Meadow Circle, PaloAlto, Calif. 94303), further described in U.S. Pat. No. 6,191,269, thedisclosure of which is incorporated herein by reference.

The THAP-family- or THAP domain polypeptide cDNA sequence is fusedin-frame by insertion of the THAP-family- or THAP domain polypeptideencoding cDNA into the SalI-BamHI site of plasmid pEGFP-NI (GenBankAccession # U55762). Cells are transiently transfected by the methodoptimal for the cell being tested (either CaPO⁴ or Lipofectin).Expression of a THAP-family or THAP domain polypeptide and induction ofapoptosis is examined using a fluorescence microscope at 24 hrs and 48hrs post-transfection. Apoptosis can be evaluated by the TUNEL method(which involves 3′ end-labeling of cleaved nuclear and/or morphologicalcriteria DNA) (Cohen et al. (1984) J. Immunol. 132:38-42, the disclosureof which is incorporated herein by reference). Where the screen uses afusion polypeptide comprising a THAP-family or THAP domain polypeptideand a reporter polypeptide (e.g., EGFP), apoptosis can be evaluated bydetection of nuclear localization of the reporter polypeptide infragmented nuclear bodies or apoptotic bodies. For example, where aTHAP-family or THAP domain polypeptide-EGFP fusion polypeptide is used,distribution of THAP-family or THAP domain polypeptide EGFP-associatedfluorescence in apoptotic cells would be identical to the distributionof DAPI or Hoechst 33342 dyes, which are conventionally used to detectthe nuclear DNA changes associated with apoptosis (Cohen et al., supra).A minimum of approximately 100 cells, which display characteristic EGFPfluorescence, are evaluated by fluorescence microscopy. Apoptosis isscored as nuclear fragmentation, marked apoptotic bodies, andcytoplasmic boiling. The characteristics of nuclear fragmentation areparticularly visible when THAP-family or THAP domain polypeptide-EGFPcondenses in apoptotic bodies.

The ability of the THAP-family- or THAP domain polypeptides to undergonuclear localization and to induce apoptosis can be tested by transientexpression in 293 human kidney cells. If proved susceptible toTHAP-family- or THAP domain- induced apoptosis, 293 cells can serve as aconvenient initial screen for those THAP family or THAP domainpolypeptides, or biologically active fragments or homologues thereofthat will likely also induce apoptosis in other (e.g. endothelial cellsor cancer cells). In an exemplary protocol, 293 cells are transfectedwith plasmid vectors expressing THAP-family- or THAP domain-EGFP fusionprotein. Approximately 5*10⁶ 293 cells in 100 mm dishes were transfectedwith 10 g of plasmid DNA using the calcium-phosphate method. Theplasmids used are comprise CMV enhancer/promoter and THAP-family- orTHAP domain-EGFP coding sequence). Apoptosis is evaluated 24 hrs aftertransfection by TUNEL and DAPI staining. The THAP-family- or THAPdomain-EGFP vector transfected cells are evaluated by fluorescencemicroscopy with observation of typical nuclear aggregation of the EGFPmarker as an indication of apoptosis. If apoptotic, the distribution ofEGFP signal in cells expressing THAP-family- or THAP domain-EGFP will beidentical to the distribution of DAPI or Hoechst 33342 dyes, which areconventionally used to detect the nuclear DNA changes associated withapoptosis (Cohen et al., supra).

The ability of the THAP family or THAP domain polypeptides, orbiologically active fragments or homologues thereof to induce apoptosiscan also be tested by expression assays in human cancer cells, forexample as available from NCI. Vector type (for example plasmid orretroviral or sindbis viral) can be selected based on efficiency in agiven cell type. After the period indicated, cells are evaluated formorphological signs of apoptosis, including aggregation of THAP-family-or THAP domain-EGFP into nuclear apoptotic bodies. Cells are countedunder a fluorescence microscope and scored as to the presence or absenceof apoptotic signs, or cells are scored by fluorescent TUNEL assay andcounted in a flow cytometer. Apoptosis is expressed as a percent ofcells displaying typical advanced changes of apoptosis.

Cells from the NCI panel of tumor cells include from example:

colon cancer, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (cell lines KM12;HT-29; SW-620; COLO205; HCT-5; HCC 2998; HCT-1 16);

CNS tumors, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (cell lines SF-268,astrocytoma; SF-539, glioblastoma; SNB-19, gliblastoma; SNB-75,astrocytoma; and U251, glioblastoma;

leukemia cells, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (cell lines CCRF-CEM,acute lymphocytic leukemia (ALL); K562, acute myelogenous leukemia(AML); MOLT-4, ALL; SR, immunoblastoma large cell; and RPMI 8226,Myeloblastoma);

prostate cancer, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (PC-3);

kidney cancer, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (cell lines 768-0; UO-31; TK10; ACHN);

skin cancer, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (Melanoma) (cell linesSKMEL-28; M14; SKMEL-5; MALME-3);

lung cancer, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (cell lines HOP-92;NCI-H460; HOP-62; NCI-H522; NCI-H23; A549; NCI-H226; EKVX; NCI-H322);

breast cancer, expression using a retroviral expression vector, withevaluation of apoptosis at 96 hrs post-infection (cell lines MCF-7;T-47D; MCF-7/ADR; MDAMB43; MDAMB23; MDA-N; BT-549);

ovary cancer, expression using either a retroviral expression vector andprotocol or the Sindbis viral expression vector and protocol, withevaluation of apoptosis at 96 hrs post-infection with retrovirus or at24 hrs post-infection with Sindbis viral vectors (cell lines OVCAR-8;OVCAR-4; IGROV-1; OVCAR-5; OVCAR3; SK-OV-3).

In a further representative example, the susceptibility of malignantmelanoma cells to apoptosis induced by a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof canbe tested in several known melanoma cell types: human melanoma WM 266-4(ATCC CRL-1676); human malignant melanoma A-375 (ATCC CRL-1619); humanmalignant, melanoma A2058 (ATCC CRL-11147); human malignant melanomaSK-MEL-31 (ATCC HTB-73); human malignant melanoma RPMI-7591 ATCC HTB-66(metastasis to lymph node). Primary melanoma isolates can also betested. In addition, human chronic myelogenous leukemia K-562 cells(ATCC CCL-243), and 293 human kidney cells (ATCC CRL-1573) (transformedprimary embryonal cell) are tested. Normal human primary dermalfibroblasts and Rat-1 fibroblasts serve as controls. All melanoma celllines are metastatic on the basis of their isolation from metastases ormetastatic nodules. A transient expression strategy is used in order toevaluate induction of a THAP-family or THAP domain polypeptide-mediatedapoptosis without artifacts associated with prolonged selection. Anexpression vector encoding the THAP-family or THAP domain polypeptide-EGFP fusion protein described below can be used in order to facilitateidentification of those cells expressing the a THAP-family or THAPdomain polypeptide. Cells are transiently transfected by the methodoptimal for the cell being tested (either CaPO₄or Lipofectin).Expression of a THAP-family or THAP domain polypeptide and induction ofapoptosis is examined using a fluorescence microscope at 24 hrs and 48hrs post-transfection. A minimum of approximately 100 cells, whichdisplay characteristic EGFP fluorescence, are evaluated by fluorescencemicroscopy. Apoptosis is scored as nuclear fragmentation, markedapoptotic bodies, and cytoplasmic boiling. The characteristics ofnuclear fragmentation are particularly visible when THAP-family or THAPdomain polypeptide-EGFP condenses in apoptotic bodies.

In a further example, the susceptibility of endothelial cells toapoptosis induced by a THAP family or THAP domain polypeptide, or abiologically active fragment or homologue thereof can be tested inseveral known endothelial cell types: HUVEC (human umbilical veinendothelial cells; BioWhittaker-Clonetics, 8830 Biggs Ford Road,Walkersville, Md. 21793-0127, Cat N° CC-2519), HMVEC-L (humanmicrovascular endothelial cells from the lung; BioWhittaker-Clonetics,8830 Biggs Ford Road, Walkersville, Md. 21793-0127, Cat N° CC-2527),HMVEC-d (human microvascular endothelial cells from the dermis;BioWhittaker-Clonetics, 8830 Biggs Ford Road, Walkersville, Md.21793-0127, Cat N° CC-2543). These and other endothelial cell types maybe useful as models in providing an indication of the ability ofTHAP-family or THAP domain polypeptides to induce apoptosis intherapeutic strategies for the regulation of angiogenesis. A transientexpression strategy is used in order to evaluate induction of aTHAP-family or THAP domain polypeptide-mediated apoptosis withoutartifacts associated with prolonged selection. An expression vectorencoding the a THAP-family or THAP domain polypeptide-EGFP fusionprotein described below can be used in order to facilitateidentification of those cells expressing the a THAP-family or THAPdomain polypeptide. Cells are transiently transfected by the methodoptimal for the cell being tested (either CaPO₄ or Lipofectin).Expression of a THAP-family or THAP domain polypeptide and induction ofapoptosis is examined using a fluorescence microscope at 24 hrs and 48hrs post-transfection. A minimum of approximately 100 cells, whichdisplay characteristic EGFP fluorescence, are evaluated by fluorescencemicroscopy. Apoptosis is scored as nuclear fragmentation, markedapoptotic bodies, and cytoplasmic boiling. The characteristics ofnuclear fragmentation are particularly visible when THAP-family or THAPdomain polypeptide-EGFP condenses in apoptotic bodies.

In another example, a transient transfection assay procedure is similarto that previously described for detecting apoptosis induced byIL-1-beta-converting enzyme (Miura et al., Cell 75: 653-660 (1993);Kumar et al., Genes Dev. 8: 1613-1626 (1994); Wang et al., Cell 78:739-750 (1994); and U.S. Pat. No. 6,221,615, the disclosures of whichare incorporated herein by reference). One day prior to transfection,cells (for example Rat-I cells) are plated in 24 well dishes at 3.5*10⁴cells/well. The following day, the cells are transfected with a markerplasmid encoding beta-galactosidase, in combination with an expressionplasmid encoding THAP-family or THAP domain polypeptide, by theLipofectamine procedure (Gibco/BRL). At 24 hours post transfection,cells are fixed and stained with X-Gal to detect beta-galactosidaseexpression in cells that received plasmid DNA (Miura et al., supra). Thenumber of blue cells is counted by microscopic examination and scored aseither live (flat blue cells) or dead (round blue cells). The cellkilling activity of the THAP-family or THAP domain polypeptide in thisassay is manifested by a large reduction in the number of blue cellsobtained relative to co-transfection of the beta-gal plasmid with acontrol expression vector (i.e., with no THAP-family or THAP domainpolypeptide cDNA insert).

In yet another example, beta-galactosidase co-transfection assays can beused for determination of cell death. The assay is performed asdescribed (Hsu, H. et al, (1995). Cell 81,495-504; Hsu, H. et al,(1996a). Cell 84, 299-308; and Hsu, H. et al, (1996b) Immunity 4,387-396 and U.S. Pat. No. 6,242,569, the disclosures of which areincorporated herein by reference). Transfected cells are stained withX-gal as described in Shu, H. B. et al, ((1995) J. Cell Sci. 108,2955-2962, the disclosure of which is incorporated herein by reference).The number of blue cells from 8 viewing fields of a 35 mm dish isdetermined by counting. The average number from one representativeexperiment is shown.

Assays for apoptosis can also be carried out by making use of anysuitable biological marker of apoptosis. Several methods are describedas follows.

In one aspect, fluorocytometric studies of cell death status can becarried out. Technology used in fluorocytometric studies employs theidentification of cells at three different phases of the cell cycle: G₁,S. and G₂. This is largely performed by DNA quantity staining bypropidium iodide labeling. Since the dying cell population contains thesame DNA quantity as the living counterparts at any of the three phasesof the cell cycle, there is no way to distinguish the two cellpopulations. One can perform double labeling for a biological marker ofapoptosis (e.g. terminin Tp30, U.S. Pat. No. 5,783,667) positivity andpropidium iodide (PI) staining together. Measurement of the labelingindices for the biological marker of apoptosis and PI staining can beused in combination to obtain the exact fractions of those cells in G₁that are living and dying. Similar estimations can be made for theS-phase and G₂ phase cell populations.

In this assay, the cells are processed for formaldehyde fixation andextraction with 0.05% Triton. Afterwards, the cell specimens areincubated with monoclonal antibody to a marker of apoptosis overnight atroom temperature or at 37C for one hour. This is followed by furtherincubation with fluoresceinated goat antimouse antibody, and subsequentincubation by propidium iodide staining. The completely processed cellspecimens are then evaluated by fluorocytometric measurement on bothfluorescence (marker of apoptosis) and rhodamine (PI) labeling intensityon a per cell basis, with the same cell population simultaneously.

In another aspect, it is possible to assess the inhibitory effect oncell growth by therapeutic induction of apoptosis. One routine method todetermine whether a particular chemotherapeutic drug can inhibitcancerous cell growth is to examine cell population size either inculture, by measuring the reduction in cell colony size or number, ormeasuring soft agar colony growth or in vivo tumor formation in nudemice, which procedures require time for development of the colonies ortumor to be large enough to be detectable. Experiments involved in theseapproaches in general require large-scale planning and multiple repeatsof lengthy experimental span (at least three weeks). Often these assaysdo not take into account the fact that a drug may not be inhibiting cellgrowth, but rather killing the cells, a more favorable consequenceneeded for chemotherapeutic treatment of cancer. Thus, assays for theassessment of apoptotis activity can involve using a biological orbiochemical marker specific for quiescent, non-cycling ornon-proliferating cells. For example, a monoclonal antibody can be usedto assess the non-proliferating population of cells in a given tissuewhich indirectly gives a measure of the proliferating component of atumor or cell mass. This detection can be combined with a biological orbiochemical marker (e.g. antibodies) to detect the dying cell populationpool, providing a powerful and rapid assessment of the effectiveness ofany given drugs in the containment of cancerous cell growth.Applications can be easily performed at the immunofluorescencemicroscopic level with cultured cells or tissue sections.

In other aspects, a biological or biochemical marker can be used toassess pharmacological intervention on inhibition of cell deathfrequency in degenerative diseases. For degenerative diseases such asAlzheimer's or Parkinson's disease, these losses may be due to thepremature activation of the cell death program in neurons. Inosteoporosis, the cell loss may be due to an improper balance betweenosteoblast and osteoclast cells, due to the too active programmed celldeath process killing more cells than the bone tissue can afford. Otherrelated phenomena may also occur in the wound healing process, tissuetransplantation and cell growth in the glomerus during kidney infection,where the balance between living and dying cell populations is anessential issue to the health status of the tissue, and are furtherdescribed in the section titled “Methods of treatment”. A rapidassessment of dying cell populations can be made through theimmunohistochemical and biochemical measurements of a biological orbiochemical marker of apoptosis in degenerative tissues. In one example,a biological or biochemical marker can be used to assess cell deathstatus in oligodendrocytes associated with Multiple Sclerosis. Positivestaining of monoclonal antibody to a marker of apoptosis (such as Tp30,U.S. Pat. No. 5,783,667, the disclosure of which is incorporated hereinby reference) occurs in dying cultured human oligodendrocytes. Theprogrammed cell death event is activated in these oligodendrocytes bytotal deprivation of serum, or by treatment with tumor necrosis factor(TNF).

In general, a biological or biochemical marker can also be used toassess cell death status in pharmacological studies in animal models.Attempting to control either a reduced cell death rate, in the case ofcancer, or an increased cell death rate, in the case ofneurodegeneration, has been recently seen as a new mode of diseaseintervention. Numerous approaches via either intervention with knowndrugs or gene therapy are in progress, starting from the base ofcorrecting the altered programmed cell death process, with the concepton maintaining a balanced cell mass in any given tissue. For thesetherapeutic interventions, the bridge between studies in cultured cellsand clinical trials is animal studies, i.e. success in intervention withanimal models, in either routine laboratory animals or transgenic micebearing either knock-out or overexpression phenotypes. Thus, abiological or biochemical marker of apoptosis, such as an antibody foran apoptosis-specific protein, is a useful tool for examining apoptoticdeath status in terms of change in dying cell numbers between normal andexperimentally manipulated animals. In this context the invention, as adiagnostic tool for assessing cell death status, could help to determinethe efficacy and potency of a drug or a gene therapeutic approach.

As discussed, provided are methods for assessing the activity ofTHAP-family members and therapeutic treatment acting on THAP-familymembers or related biological pathways. However, in other aspects, thesame methods may be used for assessment of apoptosis in general, when aTHAP-family member is used as a biological marker of apoptosis. Thus,the invention also provides diagnostic and assay methods using aTHAP-family member as a marker of cell death or apoptotic activity.Further diagnostic assays are also provided herein in the section titled‘Diagnostic and prognostic uses’.

Chemokine Binding by THAP-Family Proteins

Some embodiments of the present invention relate to THAP-familypolypeptides, chemokine-binding domains of THAP-family polypeptides,THAP oligomers, and chemokine-binding domain-THAP-immunoglobulin fusionproteins such as those described above which bind to chemokines otherthan SLC. For example, THAP-family polypeptides, chemokine-bindingdomains of THAP-family polypeptides, THAP oligomers, andchemokine-binding domain-THAP-immunoglobulin fusion proteins can be usedto bind to or otherwise interact with chemokines from many families suchas C chemokines, CC chemokines, C—X—C chemokines, C—X3-C chemokines, XCchemokines or CCK chemokines. In particular, THAP-family polypeptides,chemokine-binding domains of THAP-family polypeptides, THAP oligomers,and chemokine-binding domain-THAP-immunoglobulin fusion proteins mayinteract with chemokines such as XCL1, XCL2, CCL1, CCL2, CCL3, CCL3L1,SCYA3L2, CCL4, CCL4L, CCL5, CCL6, CCL7, CCL8, SCYA9, SCYA10, CCL1,SCYA12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, clone 391, CARP CC-1,CCL1, CK-1, regakine-1, K203, CXCL1, CXCL1P, CXCL2, CXCL3, PF4, PF4V1,CXCL5, CXCL6, PPBP, SPBPBP, IL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14,CXCL15, CXCL16, NAP-4, LFCA-1, Scyba, JSC, VHSV-induced protein, CX3CL1and fCL1.

In some embodiments of the present invention, THAP-family polypeptides,chemokine-binding domains of THAP-family polypeptides, THAP oligomers,and chemokine-binding domain-THAP-immunoglobulin fusion proteins canbind to a chemokine extracellularly. For example, the interaction of theTHAP1 polypeptide, a biologically active fragment thereof (such as thechemokine-binding domain of THAP1 (amino acids 143-213 of SEQ ID NO:3)), an oligomer thereof, or an immunoglobulin fusion thereof can bindto a chemokine extracellularly. In other examples, chemokine-bindingdomains of other THAP-family members such as THAP2, THAP3, THAP4, THAP5,THAP6, THAP7, THAP8, THAP9, THAP10, THAP11 or THAP0, biologically activefragments thereof, oligomers thereof, or immunoglobulin fusions thereofcan be used to bind to chemokines extracellularly. Binding of theTHAP-family polypeptides, chemokine-binding domains of THAP-familypolypeptides, THAP oligomers, and chemokine-bindingdomain-THAP-immunoglobulin fusion proteins may either decrease orincrease the affinity of the chemokine for its extracellular receptor.In cases where binding of the chemokine to its extracellular receptor isinhibited, the normal biological effect of the chemokine can beinhibited. Such inhibition can prevent the occurrence ofchemokine-mediated cellular responses, such as the modulation of cellproliferation, the modulation of angiogenesis, the modulation of aninflammation response, the modulation of apoptosis, the modulation ofcell differentiation. Alternatively, in cases where binding of thechemokine to its extracellular receptor is activated, the normalbiological effect of the chemokine can be enhanced. Such enhancement canincrease the occurrence of chemokine-mediated cellular responses, suchas the modulation of cell proliferation, the modulation of angiogenesis,the modulation of an inflammation response, the modulation of apoptosis,the modulation of cell differentiation.

As used herein, “ELC/CCL19”, “CCL19” and “ELC” are synonymous.

As used herein, “Rantes/CCL5”, “CCL5” and “Rantes” are synonymous.

As used herein, “MIG/CXCL9”, “CXCL9” and “MIG” are synonymous.

As used herein, “IP10/CXCL10”, “CXCL10” and “IP10” are synonymous.

In some embodiments of the present invention a chemokine-binding domainthat consists essentially of the chemokine binding portion of aTHAP-family polypeptide is contemplated. In some embodiments, theTHAP-family polypeptide is THAP-1 (SEQ ID NO: 3) or a homolog thereof.Chemokines that are capable of binding to any particular THAP-familymember can be determined as described in Examples 16, 32 and 33, whichset out both in vitro and in vivo assays for determining the bindingaffinity of several different chemokines to THAP-1. The portion of theTHAP-family protein that binds to the chemokine can readily bedetermined through the analysis of deletion and point mutants of any ofthe THAP-family members capable of chemokine-binding. Such analyses ofdeletion and point mutants were used to determine the specific region ofTHAP-1 that permits SLC-binding (see Example 15). Additionally, deletionand point mutation studies were used to determine portions ofTHAP-family proteins as well as specific amino acid residues thatinteract with PAR-4 (Examples 4-7 and 13). It will be appreciated thatthe methods described in these Examples can be used to preciselyidentify the chemokine-binding portion of any THAP-family member usingany chemokine.

By “chemokine-binding domain” or “portion that binds to a chemokine” ismeant a fragment which comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 160, 170, 180, 190, 200, 210 or greater than 210 consecutive aminoacids of a THAP-family polypeptide but less than the total number ofamino acids present in the THAP-family polypeptide. In some embodiments,the THAP-family polypeptide is THAP-1 (SEQ ID NO: 3).

The complete amino acid sequence of each human THAP-family polypeptideis described in the Sequence Listing. In particular, THAP-1 is (SEQ IDNO: 3), THAP-2 is (SEQ ID NO: 4), THAP-3 is (SEQ ID NO: 5), THAP-4 is(SEQ ID NO: 6), THAP-5 is (SEQ ID NO: 7), THAP-6 is (SEQ ID NO: 8),THAP-7 is (SEQ ID NO: 9), THAP-8 is (SEQ ID NO: 10), THAP-9 is (SEQ IDNO:11), THAP-11 is (SEQ ID NO: 12), THAP-11 is (SEQ ID NO: 13), THAP-0is (SEQ ID NO: 14). The complete amino acid sequence of additionalTHAP-family polypeptides from other species are also listed in theSequence Listing as SEQ ID NOs: 16-98. As such, the chemokine-bindingportion of any of these THAP-family polypeptide sequences that arelisted in the Sequence Listing is explicitly described. In particular,in some embodiments, the chemokine-binding domain is a fragment of aTHAP-family chemokine-binding agent described by the formula:

for each THAP-family polypeptide, N=the number of amino acids in thefull-length polypeptide; B=a number between 1 and N-1; and E=a numberbetween 1 and N.

For any THAP-family polypeptide, a chemokine-binding domain is specifiedby any consecutive sequence of amino acids beginning at an amino acidposition B and ending at amino acid position E, wherein E>B.

Methods of Complex Formation between a Chemokine and a THAP-TypeChemokine-Binding Agent

Some aspects of the present invention related to methods for forming acomplex between a chemokine and a THAP-type chemokine-binding agent.These methods include the step of contacting one or more chemokines withone or more THAP-type chemokine-binding agents described herein suchthat a complex comprising one or more chemokines and one or moreTHAP-type chemokine-binding agents is formed. In some embodiments, aplurality of different chemokines are contacted with one or a pluralityof different THAP-type chemokine-binding agents so as to form one ormore complexes. Alternatively, a plurality of different THAP-typechemokine-binding agents are contacted with one or more chemokines so asto form one or more complexes.

A number of different chemokines can be used in the above-describedcomplex formation methods. Such chemokines include, but are not limitedto, XCL1, XCL2, CCL1, CCL2, CCL3, CCL3L1, SCYA3L2, CCL4, CCL4L, CCL5,CCL6, CCL7, CCL8, SCYA9, SCYA10, CCL11, SCYA12, CCL13, CCL14, CCL15,CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25,CCL26, CCL27, CCL28, clone 391, CARP CC-1, CCL1, CK-1, regakine-1, K203,CXCL,1, CXCL1P, CXCL2, CXCL3, PF4, PF4V1, CXCL5, CXCL6, PPBP, SPBPBP,IL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14, CXCL15, CXCL16, NAP-4,LFCA-1, Scyba, JSC, VHSV-induced protein, CX3CL1 and fCL1.

Method of forming a complex between a THAP-type chemokine-binding agentand a chemokine can be used both in vitro and in vivo. For example, invitro uses can include the detection of a chemokine in a solution or abiological sample that has been removed or withdrawn from a subject.Such samples may include, but are not limited to, tissue samples, bloodsamples, and other fluid or solid samples of biological material. Invivo uses can include, but are not limited to, the detection orlocalization of chemokines in a subject, reducing or inhibiting theactivity of one or more chemokines throughout or in certain areas of asubject's body, and reducing the symptoms associated with a chemokineinfluenced or mediated condition.

Methods of Treatment

A large body of evidence gathered from experiments carried out withapoptosis modulating strategies suggests that treatments acting onapoptosis-inducing or cell proliferation-reducing proteins may offer newtreatment methods for a wide range of disorders. Methods of treatmentaccording to the invention may act in a variety of manners, given thenovel function provided for a number of proteins, and the linking ofseveral biological pathways.

Provided herein are treatment methods based on the functionalization ofthe THAP-family members. THAP family or THAP domain polypeptides, andbiologically active fragments and homologues thereof, as describedfurther herein may be useful in modulation of apoptosis or cellproliferation.

The methods of treatment involve acting on a molecule of the invention(that is, a THAP family member polypeptide, THAP-family target, or PAR4or PAR4 target). Included are methods which involve modulatingTHAP-family polypeptide activity, THAP-family target activity, or PAR4or PAR4 target activity. This modulation (increasing or decreasing) ofactivity can be carried out in a number of suitable ways, several ofwhich have been described in the present application.

For example, methods of treatment may involve modulating a “THAP-familyactivity”, “biological activity of a THAP-family member” or “functionalactivity of a THAP-family member”. Modulating THAP-family activity mayinvolve modulating an association with a THAP-family-target molecule(for example, association of THAP1, THAP2 or THAP3 with Par4 orassociation of THAP 1, THAP2 or THAP3 with a PML-NB protein) orpreferably any other activity selected from the group consisting of: (1)mediating apoptosis or cell proliferation when expressed or introducedinto a cell, most preferably inducing or enhancing apoptosis, and/ormost preferably reducing cell proliferation; (2) mediating apoptosis orcell proliferation of an endothelial cell; (3) mediating apoptosis orcell proliferation of a hyperproliferative cell; (4) mediating apoptosisor cell proliferation of a CNS cell, preferably a neuronal or glialcell; or (5) an activity determined in an animal selected from the groupconsisting of mediating, preferably inhibiting angiogenesis, mediating,preferably inhibiting inflammation, inhibition of metastatic potentialof cancerous tissue, reduction of tumor burden, increase in sensitivityto chemotherapy or radiotherapy, killing a cancer cell, inhibition ofthe growth of a cancer cell, or induction of tumor regression. DetectingTHAP-family activity may also comprise detecting any suitabletherapeutic endpoint associated with a disease condition discussedherein.

In another example, methods of treatment may involve modulating a “PAR4activity”, “biological activity of PAR4” or “functional activity ofPAR4”. Modulating PAR4 activity may involve modulating an associationwith a PAR4-target molecule (for example THAP1, THAP2, THAP3 or PML-NBprotein) or most preferably PAR4 apoptosis inducing or enhancing (e.g.signal transducing) activity, or inhibition of cell proliferation orcell cycle.

Methods of treatment may involve modulating the recruitment, binding orassociation of proteins to PML-NBs, or otherwise modulating PML-NBsactivity. The present invention also provides methods for modulatingPAR4 activity, comprising modulating PAR4 interactions with THAP-familyproteins, and PAR4 and PML-NBs, as well as modulating THAP-familyactivity, comprising modulating for example THAP1 interactions withPML-NBs. The invention encompasses inhibiting or increasing therecruitment of THAP1, or PAR4 to PML-NBs. Preventing the binding ofeither or both of THAP1 or PAR4 to PML-NBs may increase thebioavailability or THAP1 and/or PAR4, thus providing a method ofincreasing THAP1 and/or PAR4 activity. The invention also encompassesinhibiting or increasing the binding of a THAP-family protein (such asTHAP1) or PAR4 to PML-NBs or to another protein associated with PML-NBs,such as a protein selected from the group consisting of daxx, sp100,sp140, p53, pRB, CBP, BLM, SUMO-1. For example, the inventionencompasses modulating PAR4 activity by preventing the binding of THAP1to PAR4, or by preventing the recruitment or binding of PAR4 to PML-NBs.

Therapeutic methods and compositions of the invention may involve (1)modulating apoptosis or cell proliferation, most preferably inducing orenhancing apoptosis, and/or most preferably reducing cell proliferation;(2) modulating apoptosis or cell proliferation of an endothelial cell(3) modulating apoptosis or cell proliferation of a hyperproliferativecell; (4) modulating apoptosis or cell proliferation of a CNS cell,preferably a neuronal or glial cell; (5) inhibition of metastaticpotential of cancerous tissue, reduction of tumor burden, increase insensitivity to chemotherapy or radiotherapy, killing a cancer cell,inhibition of the growth of a cancer cell, or induction tumorregression; or (6) interaction with a THAP family target molecule orTHAP domain target molecule, preferably interaction with a protein or anucleic acid. Methods may also involve improving a symptom of orameliorating a condition as further described herein.

Antiapoptotic Therapy

Molecules of the invention (e.g. those obtained using the screeningmethods described herein, dominant negative mutants, antibodies etc.)which inhibit apoptosis are also expected to be useful in the treatmentand/or prevention of disease. Diseases in which it is desirable toprevent apoptosis include neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa and cerebellar degeneration; myelodysplasis such as aplasticanemia; ischemic diseases such as myocardial infarction and stroke;hepatic diseases such as alcoholic hepatitis, hepatitis B and hepatitisC; joint-diseases such as osteoarthritis; atherosclerosis; and etc. Theapoptosis inhibitor of the present invention is especially preferablyused as an agent for prophylaxis or treatment of a neurodegenerativedisease (see also Adams, J. M., Science, 281:1322 (1998).

Included as inhibitors of apoptosis as described herein are generallyany molecule which inhibits activity of a THAP family or THAP domainpolypeptide, or a biologically active fragment or homologue thereof, aTHAP-family target protein or PAR4 (particularly PAR4/PML-NB proteininteractions). THAP-family and THAP domain polypeptides inhibitors mayinclude for example antibodies, peptides, dominant negative THAP-familyor THAP domain analogs, small molecules, ribozyme or antisense nucleicacids. These inhibitors may be particularly advantageous in thetreatment of neurodegenerative disorders. Particularly preferred areinhibitors which affect binding of THAP-family protein to a THAP-familytarget protein, and inhibitors which affect the DNA binding activity ofa THAP-family protein.

In further preferred aspects the invention provides inhibitors ofTHAP-family activity, including but not limited to molecules whichinterfere or inhibit interactions of THAP-family proteins with PAR4, forthe treatment of endothelial cell related disorders andneurodegenerative disorders. Support is found in the literature, as PAR4appears to play a key role in neuronal apoptosis in variousneurodegenerative disorders (Guo et al., 1998; Mattson et al., 2000;Mattson et al., 1999; Mattson et al., 2001). THAP1, which is expressedin brain and associates with PAR4 may therefore also play a key role inneuronal apoptosis. Drugs that inhibit THAP-family and/or inhibitTHAP-family/PAR4 complex formation may lead to the development of novelpreventative and therapeutic strategies for neurodegenerative disorders.

Apoptosis Regulation in Endothelial Cells

The invention also provides methods of regulating angiogenesis in asubject which are expected to be useful in the treatment of cancer,cardiovascular diseases and inflammatory diseases. An inducer ofapoptosis of immortalized cells is expected to be useful in suppressingtumorigenesis and/or metastasis in malignant tumors. Examples ofmalignant tumors include leukemia (for example, myelocytic leukemia,lymphocytic leukemia such as Burkitt lymphoma), digestive tractcarcinoma, lung carcinoma, pancreas carcinoma, ovary carcinoma, uteruscarcinoma, brain tumor, malignant melanoma, other carcinomas, andsarcomas. The present inventors have isolated both THAP1 and PAR4 cDNAsfrom human endothelial cells, and both PAR4 and PML are known to beexpressed predominantly in blood vessel endothelial cells (Boghaert etal., (1997) Cell Growth Differ 8(8):881-90; Terris B. et al, (1995)Cancer Res. 55(7):1590-7, 1995,.the disclosures of which areincorporated herein by reference), suggesting that the PML-NBs-and thenewly associated THAP1/PAR4 proapoptotic complex may be a majorregulator of endothelial cell apoptosis in vivo and thus constitute anattractive therapeutic target for angiogenesis-dependent diseases. Forexample, THAP1 and PAR4 pathways may allow selective treatments thatregulate (e.g. stimulate or inhibit) angiogenesis.

In a first aspect, the invention provides methods of inhibitingendothelial cell apoptosis, by administering a THAP1 or PAR4 inhibitor,or optionally a THAP1/PAR4 interaction inhibitor or optionally aninhibitor of THAP1 DNA binding activity. As further described herein,the THAP domain is involved in THAP1 pro-apoptotic activity. Deletion ofthe THAP domain abrogates the proapoptotic activity of THAP1 in mouse3T3 fibroblasts, as shown in Example 11. Also, as further describedherein, deletion of residues 168-172 or replacement of residues 171-172abrogates THAP1 binding to PAR4 both in vitro and in vivo and results inlack of recruitment of PAR4 by THAP1 to PML-NBs. For PAR4, the leucinezipper domain is required (and is sufficient) for binding to THAP1.

Inhibiting endothelial cell apoptosis may improve angiogenesis andvasculogenesis in patients with ischemia and may also interfere withfocal dysregulated vascular remodeling, the key mechanism foratherosclerotic disease progression.

In another aspect, the invention provides methods of inducingendothelial cell apoptosis, by administering for example a biologicallyactive THAP family polypeptide such as THAP1, a THAP domain polypeptideor a PAR4 polypeptide, or a biologically active fragment or homologuethereof, or a THAP1 or PAR4 stimulator. Stimulation of endothelial cellapoptosis may prevent or inhibit angiogenesis and thus limit unwantedneovascularization of tumors or inflamed tissues (see Dimmeler andZeiher, Circulation Research, 2000, 87:434-439, the disclosure of whichis incorporated herein by reference).

Angiogenesis

Angiogenesis is defined in adult organism as the formation of new bloodvessels by a process of sprouting from pre-existing vessels. Thisneovascularization involves activation, migration, and proliferation ofendothelial cells and is driven by several stimuli, among those shearstress. Under normal physiological conditions, humans or animals undergoangiogenesis only in very specific restricted situations. For example,angiogenesis is normally observed in wound healing, fetal and embryonaldevelopment and formation of the corpus luteum, endometrium andplacenta. Molecules of the invention may have endothelial inhibiting orinducing activity, having the capability to inhibit or induceangiogenesis in general.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, tumor metastasis and abnormal growth by endothelial cells andsupports the pathological damage seen in these conditions. The diversepathological disease states in which unregulated angiogenesis is presenthave been grouped together as angiogenic dependent or angiogenicassociated diseases. It is thus an object of the present invention toprovide methods and compositions for treating diseases and processesthat are mediated by angiogenesis including, but not limited to,hemangioma, solid tumors, leukemia, metastasis, telangiectasia psoriasisscleroderma, pyogenic granuloma, Myocardial angiogenesis, plaqueneovascularization, cororany collaterals, ischemic limb angiogenesis,corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, wound healing, peptic ulcer, fractures, keloids,vasculogenesis, hematopoiesis, ovulation, menstruation, andplacentation.

(i) Anti-Angiogenic Therapy

In one aspect the invention provides anti-angiogenic therapies aspotential treatments for a wide variety of diseases, including cancer,arteriosclerosis, obesity, arthritis, duodenal ulcers, psoriasis,proliferative skin disorders, cardiovascular disorders and abnormalocular neovascularization caused, for example, by diabetes (Folkman,Nature Medicine 1:27 (1995) and Folkman, Seminars in Medicine of theBeth Israel Hospital, Boston, New England Journal of Medicine, 333:1757(1995)). Anti-angiogenic therapies are thought to act by inhibiting theformation of new blood vessels.

The present invention thus provides methods and compositions fortreating diseases and processes mediated by undesired and uncontrolledangiogenesis by administering to a human or animal a compositioncomprising a substantially purified THAP family or THAP domainpolypeptide, or a biologically active fragment, homologue or derivativethereof in a dosage sufficient to inhibit angiogenesis, administering avector capable of expressing a nucleic acid encoding a THAP-family orTHAP domain protein, or administering any other inducer of expression oractivity of a THAP-family or THAP domain protein. The present inventionis particularly useful for treating or for repressing the growth oftumors. Administration of THAP-family or THAP domain nucleic acid,protein or other inducer to a human or animal with prevascularizedmetastasized tumors will prevent the growth or expansion of thosetumors. THAP-family activity may be used in combination with othercompositions and procedures for the treatment of diseases. For example,a tumor may be treated conventionally with surgery, radiation orchemotherapy combined with THAP-family or THAP domain protein and thenTHAP-family or THAP domain protein may be subsequently administered tothe patient to extend the dormancy of micrometastases and to stabilizeany residual primary tumor.

In a preferred example, a THAP-family polypeptide activity, preferably aTHAP1 activity is used for the treatment of arthritis, for examplerheumatiod arthritis. Rheumatoid arthritis is characterized bysymmetric, polyarticular inflammation of synovial-lined joints, and mayinvolve extraarticular tissues, such as the pericardium, lung, and bloodvessels.

(ii) Angiogenic Therapy

In another aspect, the inhibitors of THAP-family protein activity,particularly THAP1 activity, could be used as an anti-apoptotic and thusas an angiogenic therapy. Angiogenic therapies are potential treatmentsfor promoting wound healing and for stimulating the growth of new bloodvessels to by-pass occluded ones. Thus, pro-angiogenic therapies couldpotentially augment or replace by-pass surgeries and balloon angioplasty(PTCA). For example, with respect to neovascularization to bypassoccluded blood vessels, a “therapeutically effective amount” is aquantity which results in the formation of new blood vessels which cantransport at least some of the blood which normally would pass throughthe blocked vessel.

The THAP-family protein of the present invention can for example be usedto generate antibodies that can be used as inhibitors of apoptosis. Theantibodies can be either polyclonal antibodies or monoclonal antibodies.In addition, these antibodies that specifically bind to the THAP-familyprotein can be used in diagnostic methods and kits that are well knownto those of ordinary skill in the art to detect or quantify theTHAP-family protein in a body fluid. Results from these tests can beused to diagnose or predict the occurrence or recurrence of a cancer andother angiogenic mediated diseases.

It will be appreciated that other inhibitors of THAP-family and THAPdomain proteins can also be used in angiogenic therapies, including forexample small molecules, antisense nucleic acids, dominant negativeTHAP-family and THAP domain proteins or peptides identified using theabove methods.

In view of applications in both angiogenic and antiangiogenic therapies,molecules of the invention may have endothelial inhibiting or inducingactivity, having the capability to inhibit or induce angiogenesis ingeneral. It will be appreciated that methods of assessing suchcapability are known in the art, including for example assessingantiangiogenic properties as the ability inhibit the growth of bovinecapillary endothelial cells in culture in the presence of fibroblastgrowth factor.

It is to be understood that the present invention is contemplated toinclude any derivatives of the THAP family or THAP domain polypeptides,and biologically active fragments and homologues thereof that haveendothelial inhibitory or apoptotic activity. The present inventionincludes full-length THAP-family and THAP domain proteins, derivativesof the THAP-family and THAP domain proteins and biologically-activefragments of the THAP-family and THAP domain proteins. These includeproteins with THAP-family protein activity that have amino acidsubstitutions or have sugars or other molecules attached to amino acidfunctional groups. The methods also contemplate the use of genes thatcode for a THAP-family protein and to proteins that are expressed bythose genes.

As discussed, several methods are described herein for delivering amodulator to a subject in need of treatment, including for example smallmolecule modulators, nucleic acids including via gene therapy vectors,and polypeptides including peptide mimetics, active polypeptides,dominant negative polypeptides and antibodies. It will be thus beappreciated that modulators of the invention identified according to themethods in the section titled “Drug Screening Assays” can be furthertested in cell or animal models for their ability to ameliorate orprevent a condition involving a THAP-family polypeptide, particularlyTHAP1, THAP1, THAP2 or THAP3/PAR4 interactions, THAP-family DNA bindingor PAR4/PML-NBs interactions. Likewise, nucleic acids, polypeptides andvectors (e.g. viral) can also be assessed in a similar manner.

An “individual” treated by the methods of this invention is avertebrate, particularly a mammal (including model animals of humandisease, farm animals, sport animals, and pets), and typically a human.“Individual” is also synonymous with “subject.”

“Treatment” refers to clinical intervention in an attempt to alter thenatural course of the individual being treated, and may be performedeither for prophylaxis or during the course of clinical pathology.Desirable effects include preventing occurrence or recurrence ofdisease, alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, such as hyperresponsiveness,inflammation, or necrosis, lowering the rate of disease progression,amelioration or palliation of the disease state, and remission orimproved prognosis. The “pathology” associated with a disease conditionis anything that compromises the well-being, normal physiology, orquality of life of the affected individual.

Treatment is performed by administering an effective amount of aTHAP-family polypeptide inhibitor or activator. An “effective amount” isan amount sufficient to effect a beneficial or desired clinical result,and can be administered in one or more doses. The criteria for assessingresponse to therapeutic modalities employing the lipid compositions ofthis invention are dictated by the specific condition, measuredaccording to standard medical procedures appropriate for the condition.

Reducing Chemokine Mediated Effects

Some aspects of the present invention relate to the use of THAP-familypolypeptides, including THAP-1, chemokine-binding domains of THAP-familypolypeptides, THAP-family polypeptide or THAP-family chemokine-bindingdomain fusions to immunoglobulin Fc, oligomers of THAP-familypolypeptides or THAP-family chemokine-binding domains, or homologs ofany of the above-listed compositions (together and herein after referredto as THAP-type chemokine-binding agents) for reducing the inflammationor the symptoms associated with diseases or conditions that areinfluenced or mediated by chemokine binding or activity. In suchembodiments, the THAP-type chemokine binding agents are administered toa subject in effective amounts so as to reduce the symptoms associatedwith the condition. In some embodiments, the chemokine that is effectedby the THAP-type chemokine binding agent is SLC, CCL19, CCL5, CXCL9,CXCL10 or a combination of these chemokines. In other embodiments, thechemokine that is effected by the THAP-type chemokine binding agent isXCL1, XCL2, CCL1, CCL2, CCL3, CCL3L1, SCYA3L2, CCL4, CCL4L, CCL5, CCL6,CCL7, CCL8, SCYA9, SCYA10, CCL11, SCYA12, CCL13, CCL14, CCL15, CCL16,CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26,CCL27, CCL28, clone 391, CARP CC-1, CCL1, CK-1, regakine-1, K203, CXCL1,CXCL1P, CXCL2, CXCL3, PF4, PF4V1, CXCL5, CXCL6, PPBP, SPBPBP, IL8,CXCL9, CXCL10, CXCL11, CXCL12, CXCL14, CXCL15, CXCL16, NAP-4, LFCA-1,Scyba, JSC, VHSV-induced protein, CX3CL1, fCL1 or a combination of thesechemokines. In some embodiments, the THAP-type chemokine-binding agentis administered directly whereas in other embodiments it is administeredas a pharmaceutical composition. In either case, the routes ofadministration that are known in the art and described herein may beused to deliver the THAP-type chemokine-binding agent to the subject.

Some embodiments of the present invention relate to a device fordelivering the THAP-type chemokine-binding agent or pharmaceuticalcomposition thereof to the subject. In such embodiment, the devicecomprises a container which contains the THAP-type chemokine-bindingagent or pharmaceutical composition thereof. For example, in someembodiments, the device may be a conventional device including, but notlimited to, syringes, devices for intranasal administration ofcompositions and vaccine guns. In one embodiment, the device comprises amember which receives the THAP-type chemokine-binding agent orpharmaceutical composition thereof in communication with a mechanism fordelivering the composition to the subject. In some embodiments, thedevice is an inhaler or a patch for transdermal administration.

Pharmaceutical Compositions

Compounds capable of inhibiting THAP-family activity, preferably smallmolecules but also including peptides, THAP-family nucleic acidmolecules, THAP-family proteins, and anti-THAP-family antibodies (alsoreferred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELα (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifumgal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Where the active compound is a protein, peptide or anti-THAP-familyantibody, sterile injectable solutions can be prepared by incorporatingthe active compound (e.g.,) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. For administration by inhalation, thecompounds are delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. Systemic administration can alsobe by transmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. Most preferably, active compound is delivered to a subject byintravenous injection.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, the disclosure of which is incorporated herein by referencein its entirety.

It is especially advantageous to formulate oral or preferably parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

It will be appreciated that THAP-type chemokine-binding agents can beformulated as pharmaceutical compositions and administered as describedabove. Additionally, the effective dose, route of administration,duration of administration, duration between doses and therapeuticeffect can be determined by the methods described above as well as usingmethods that are well known in the art.

Diagnostic and Prognostic Uses

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenetics; and in drug screening and methods of treatment (e.g.,therapeutic and prophylactic) as further described herein.

The invention provides diagnostic and prognositc assays for detectingTHAP-family members, as further described. Also provided are diagnosticand prognostic assays for detecting interactions between THAP-familymembers and THAP-family target molecules. In a preferred example, aTHAP-family member is THAP1, THAP2 or THAP3 and the THAP-family targetis PAR4 or a PML-NB protein.

The invention also provides diagnostic and prognositc assays fordetecting THAP1 and/or PAR4 localization to or association with PML-NBs,or association with or binding to a PML-NB-associated protein, such asdaxx, sp100, sp140, p53, pRB, CBP, BLM or SUMO-1. In a preferred method,the invention provides detecting PAR4 localization to or associationwith PML-NBs. In a further aspect, the invention provides detectingTHAP-family nucleic acid binding activity.

The isolated nucleic acid molecules of the invention can be used, forexample, to detect THAP-family polypeptide mRNA (e.g., in a biologicalsample) or a genetic alteration in a THAP-family gene, and to modulate aTHAP-family polypeptide activity, as described further below. TheTHAP-family proteins can be used to treat disorders characterized byinsufficient or excessive production of a THAP-family protein orTHAP-family target molecules. In addition, the THAP-family proteins canbe used to screen for naturally occurring THAP-family target molecules,to screen for drugs or compounds which modulate, preferably inhibitTHAP-family activity, as well as to treat disorders characterized byinsufficient or excessive production of THAP-family protein orproduction of THAP-family protein forms which have decreased or aberrantactivity compared to THAP-family wild type protein. Moreover, theanti-THAP-family antibodies of the invention can be used to detect andisolate THAP-family proteins, regulate the bioavailability ofTHAP-family proteins, and modulate THAP-family activity.

Accordingly one embodiment of the present invention involves a method ofuse (e.g., a diagnostic assay, prognostic assay, or aprophylactic/therapeutic method of treatment) wherein a molecule of thepresent invention (e.g., a THAP-family protein, THAP-family nucleicacid, or most preferably a THAP-family inhibitor or activator) is used,for example, to diagnose, prognose and/or treat a disease and/orcondition in which any of the aforementioned THAP-family activities isindicated. In another embodiment, the present invention involves amethod of use (e.g., a diagnostic assay, prognostic assay, or aprophylactic/therapeutic method of treatment) wherein a molecule of thepresent invention (e.g., a THAP-family protein, THAP-family nucleicacid, or a THAP-family inhibitor or activator) is used, for example, forthe diagnosis, prognosis, and/or treatment of subjects, preferably ahuman subject, in which any of the aforementioned activities ispathologically perturbed. In a preferred embodiment, the methods of use(e.g., diagnostic assays, prognostic assays, or prophylactic/therapeuticmethods of treatment) involve administering to a subject, preferably ahuman subject, a molecule of the present invention (e.g., a THAP-familyprotein, THAP-family nucleic acid, or a THAP-family inhibitor oractivator) for the diagnosis, prognosis, and/or therapeutic treatment.In another embodiment, the methods of use (e.g., diagnostic assays,prognostic assays, or prophylactic/therapeutic methods of treatment)involve administering to a human subject a molecule of the presentinvention (e.g., a THAP-family protein, THAP-family nucleic acid, or aTHAP-family inhibitor or activator).

For example, the invention encompasses a method of determining whether aTHAP-family member is expressed within a biological sample comprising:a) contacting said biological sample with: ii) a polynucleotide thathybridizes under stringent conditions to a THAP-family nucleic acid; oriii) a detectable polypeptide (e.g. antibody) that selectively binds toa THAP-family polypeptide; and b) detecting the presence or absence ofhybridization between said polynucleotide and an RNA species within saidsample, or the presence or absence of binding of said detectablepolypeptide to a polypeptide within said sample. A detection of saidhybridization or of said binding indicates that said THAP-family memberis expressed within said sample. Preferably, the polynucleotide is aprimer, and wherein said hybridization is detected by detecting thepresence of an amplification product comprising said primer sequence, orthe detectable polypeptide is an antibody.

Also envisioned is a method of determining whether a mammal, preferablyhuman, has an elevated or reduced level of expression of a THAP-familymember, comprising: a) providing a biological sample from said mammal;and b) comparing the amount of a THAP-family polypeptide or of aTHAP-family RNA species encoding a THAP-family polypeptide within saidbiological sample with a level detected in or expected from a controlsample. An increased amount of said THAP-family polypeptide or saidTHAP-family RNA species within said biological sample compared to saidlevel detected in or expected from said control sample indicates thatsaid mammal has an elevated level of THAP-family expression, and adecreased amount of said THAP-family polypeptide or said THAP-family RNAspecies within said biological sample compared to said level detected inor expected from said control sample indicates that said mammal has areduced level of expression of a THAP-family member.

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining THAP-familyprotein and/or nucleic acid expression as well as THAP-family activity,in the context of a biological sample (e.g., blood, serum, cells,tissue) to thereby determine whether an individual is afflicted with adisease or disorder, or is at risk of developing a disorder, associatedwith aberrant THAP-family expression or activity. The invention alsoprovides for prognostic (or predictive) assays for determining whetheran individual is at risk of developing a disorder associated with aTHAP-family protein, nucleic acid expression or activity. For example,mutations in a THAP-family gene can be assayed in a biological sample.Such assays can be used for prognostic or predictive purpose to therebyphophylactically treat an individual prior to the onset of a disordercharacterized by or associated with a THAP-family protein, nucleic acidexpression or activity.

Accordingly, the methods of the present invention are applicablegenerally to diseases related to regulation of apoptosis, including butnot limited to disorders characterized by unwanted cell proliferation orgenerally aberrant control of differentiation, for example neoplastic orhyperplastic disorders, as well as disorders related to proliferation orlack thereof of endothelial cells, inflammatory disorders andneurodegenerative disorders.

Diagnostic Assays

An exemplary method for detecting the presence (quantitative or not) orabsence of a THAP-family protein or nucleic acid in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting a THAP-family protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes THAP-family protein such that the presence of theTHAP-family protein or nucleic acid is detected in the biologicalsample. A preferred agent for detecting a THAP-family mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to aTHAP-family mRNA or genomic DNA. The nucleic acid probe can be, forexample, a full-length THAP-family nucleic acid, such as the nucleicacid of SEQ ID NO: 160 such as a nucleic acid of at least 15, 30, 50,100, 250, 400, 500 or 1000 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to a THAP-family mRNAor genomic DNA or a portion of a THAP-family nucleic acid. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

In preferred embodiments, the subject method can be characterized bygenerally comprising detecting, in a tissue sample of the subject (e.g.a human patient), the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding oneof the subject THAP-family proteins or (ii) the mis-expression of aTHAP-family gene. To illustrate, such genetic lesions can be detected byascertaining the existence of at least one of (i) a deletion of one ormore nucleotides from a THAP-family gene, (ii) an addition of one ormore nucleotides to such a THAP-family gene, (iii) a substitution of oneor more nucleotides of a THAP-family gene, (iv) a gross chromosomalrearrangement or amplification of a THAP-family gene, (v) a grossalteration in the level of a messenger RNA transcript of a THAP-familygene, (vi) aberrant modification of a THAP-family gene, such as of themethylation pattern of the genomic DNA, (vii) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a THAP-familygene, and (viii) a non-wild type level of a THAP-family-target protein.

A preferred agent for detecting a THAP-family protein is an antibodycapable of binding to a THAP-family protein, preferably an antibody witha detectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect a THAP-familymRNA, protein, or genomic DNA in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of a THAP-familymRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of a THAP-family protein include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of a THAP-family genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of a THAP-family proteininclude introducing into a subject a labeled anti-THAP-family antibody.For example, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In yet another exemplary embodiment, aberrant methylation patterns of aTHAP-family gene can be detected by digesting genomic DNA from a patientsample with one or more restriction endonucleases that are sensitive tomethylation and for which recognition sites exist in the THAP-familygene (including in the flanking and intronic sequences). See, forexample, Buiting et al. (1994) Human Mol Genet 3:893-895. Digested DNAis separated by gel electrophoresis, and hybridized with probes derivedfrom, for example, genomic or cDNA sequences. The methylation status ofthe THAP-family gene can be determined by comparison of the restrictionpattern generated from the sample DNA with that for a standard of knownmethylation.

Furthermore, gene constructs such as those described herein can beutilized in diagnostic assays to determine if a cell's growth ordifferentiation state is no longer dependent on the regulatory functionof a THAP-family protein, e.g. in determining the phenotype of atransformed cell. Such knowledge can have both prognostic andtherapeutic benefits. To illustrate, a sample of cells from the tissuecan be obtained from a patient and dispersed in appropriate cell culturemedia, a portion of the cells in the sample can be caused to express arecombinant THAP-family protein or a THAP-family target protein, e.g. bytransfection with a expression vector described herein, or to increasethe expression or activity of an endogenous THAP-family protein orTHAP-family target protein, and subsequent growth of the cells assessed.The absence of a change in phenotype of the cells despite expression ofthe THAP-family or THAP-family target protein may be indicative of alack of dependence on cell regulatory pathways which includes theTHAP-family or THAP-family target protein, e.g. THAP-family- orTHAP-family target-mediated transcription. Depending on the nature ofthe tissue of interest, the sample can be in the form of cells isolatedfrom, for example, a blood sample, an exfoliated cell sample, a fineneedle aspirant sample, or a biopsied tissue sample. Where the initialsample is a solid mass, the tissue sample can be minced or otherwisedispersed so that cells can be cultured, as is known in the art.

In yet another embodiment, a diagnostic assay is provided which detectsthe ability of a THAP-family gene product, e.g., isolated from abiopsied cell, to bind to other cellular proteins. For instance, it willbe desirable to detect THAP-family mutants which, while expressed atappreciable levels in the cell, are defective at binding a THAP-familytarget protein (having either diminished or enhanced binding affinity).Such mutants may arise, for example, from mutations, e.g., pointmutants, which may be impractical to detect by the diagnostic DNAsequencing techniques or by the immunoassays described above. Thepresent invention accordingly further contemplates diagnostic screeningassays which generally comprise cloning one or more THAP-family genesfrom the sample cells, and expressing the cloned genes under conditionswhich permit detection of an interaction between that recombinant geneproduct and a target protein, e.g., for example the THAP1 gene and atarget PAR4 protein or a PML-NB protein. As will be apparent from thedescription of the various drug screening assays set forth below, a widevariety of techniques can be used to determine the ability of aTHAP-family protein to bind to other cellular components. Thesetechniques can be used to detect mutations in a THAP-family gene whichgive rise to mutant proteins with a higher or lower binding affinity fora THAP-family target protein relative to the wild-type THAP-family.Conversely, by switching which of the THAP-family target protein andTHAP-family protein is the “bait” and which is derived from the patientsample, the subject assay can also be used to detect THAP-family targetprotein mutants which have a higher or lower binding affinity for aTHAP-family protein relative to a wild type form of that THAP-familytarget protein.

In an exemplary embodiment, a PAR4 or a PMB-NB protein (e.g. wild-type)can be provided as an immobilized protein (a “target”), such as by useof GST fusion proteins and glutathione treated microtitre plates. ATHAP1 gene (a “sample” gene) is amplified from cells of a patientsample, e.g., by PCR, ligated into an expression vector, and transformedinto an appropriate host cell. The recombinantly produced THAP1 proteinis then contacted with the immobilized PAR4 or PMB-NB protein, e.g., asa lysate or a semi-purified preparation, the complex washed, and theamount of PAR4 or PMB-NB protein/THAP1 complex determined and comparedto a level of wild-type complex formed in a control. Detection can beby, for instance, an immunoassay using antibodies against the wild-typeform of the THAP1 protein, or by virtue of a label provided by cloningthe sample THAP1 gene into a vector which provides the protein as afusion protein including a detectable tag. For example, a myc epitopecan be provided as part of a fusion protein with the sample THAP1 gene.Such fusion proteins can, in addition to providing a detectable label,also permit purification of the sample THAP1 protein from the lysateprior to application to the immobilized target. In yet anotherembodiment of the subject screening assay, the two hybrid assay,described in the appended examples, can be used to detect mutations ineither a THAP-family gene or THAP-family target gene which alter complexformation between those two proteins.

Accordingly, the present invention provides a convenient method fordetecting mutants of THAP-family genes encoding proteins which areunable to physically interact with a THAP-family target “bait” protein,which method relies on detecting the reconstitution of a transcriptionalactivator in a THAP-family/THAP-family target-dependent fashion.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject. In another embodiment, the methodsfurther involve obtaining a control biological sample from a controlsubject, contacting the control sample with a compound or agent capableof detecting a THAP-family protein, mRNA, or genomic DNA, such that thepresence of a THAP-family protein, mRNA or genomic DNA is detected inthe biological sample, and comparing the presence of a THAP-familyprotein, mRNA or genomic DNA in the control sample with the presence ofa THAP-family protein, mRNA or genomic DNA in the test sample. Theinvention also encompasses kits for detecting the presence ofTHAP-family protein, mRNA or genomic DNA in a biological sample. Forexample, the kit can comprise a labeled compound or agent capable ofdetecting a THAP-family protein or mRNA or genomic DNA in a biologicalsample; means for determining the amount of a THAP-family member in thesample; and means for comparing the amount of THAP-family member in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect THAP-family protein or nucleic acid.

In certain embodiments, detection involves the use of a probe/primer ina polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195and 4,683,202, the disclosures of which are incorporated herein byreference in their entireties), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegrenet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364, the disclosures of which are incorporated herein byreference in their entireties), the latter of which can be particularlyuseful for detecting point mutations in the THAP-family-gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682, the disclosure ofwhich is incorporated herein by reference in its entirety). This methodcan include the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a THAP-family gene under conditions suchthat hybridization and amplification of the THAP-family-gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Genotyping assays for diagnostics generally require the previousamplification of the DNA region carrying the biallelic marker ofinterest. However, ultrasensitive detection methods which do not requireamplification are also available. Methods well-known to those skilled inthe art that can be used to detect biallelic polymorphisms includemethods such as, conventional dot blot analyzes, single strandconformational polymorphism analysis (SSCP) described by Orita et al.,PNAS 86: 2766-2770 (1989), the disclosure of which is incorporatedherein by reference in its entirety, denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis, mismatch cleavagedetection, and other conventional techniques as described in Sheffieldet al. (1991), White et al. (1992), and Grompe et al. (1989 and 1993)(Sheffield, V. C. et al, Proc. Natl. Acad. Sci. U.S.A 49:699-706 (1991);White, M. B. et al., Genomics 12:301-306 (1992); Grompe, M. et al.,Proc. Natl. Acad. Sci. U.S.A 86:5855-5892 (1989); and Grompe, M. NatureGenetics 5:111-117 (1993), the disclosures of which are incorporatedherein by reference in their entireties). Another method for determiningthe identity of the nucleotide present at a particular polymorphic siteemploys a specialized exonuclease-resistant nucleotide derivative asdescribed in U.S. Pat. No. 4,656,127, the disclosure of which isincorporated herein by reference in its entirety. Further methods aredescribed as follows.

The nucleotide present at a polymorphic site can be determined bysequencing methods. In a preferred embodiment, DNA samples are subjectedto PCR amplification before sequencing as described above. DNAsequencing methods are described in “Sequencing Of Amplified Genomic DNAAnd Identification Of Single Nucleotide Polymorphisms”. Preferably, theamplified DNA is subjected to automated dideoxy terminator sequencingreactions using a dye-primer cycle sequencing protocol. Sequenceanalysis allows the identification of the base present at the biallelicmarker site.

In microsequencing methods, the nucleotide at a polymorphic site in atarget DNA is detected by a single nucleotide primer extension reaction.This method involves appropriate microsequencing primers which,hybridize just upstream of the polymorphic base of interest in thetarget nucleic acid. A polymerase is used to specifically extend the 3′end of the primer with one single ddNTP (chain terminator) complementaryto the nucleotide at the polymorphic site. Next the identity of theincorporated nucleotide is determined in any suitable way. Typically,microsequencing reactions are carried out using fluorescent ddNTPs andthe extended microsequencing primers are analyzed by electrophoresis onABI 377 sequencing machines to determine the identity of theincorporated nucleotide as described in EP 412 883, the disclosure ofwhich is incorporated herein by reference in its entirety. Alternativelycapillary electrophoresis can be used in order to process a highernumber of assays simultaneously. Different approaches can be used forthe labeling and detection of ddNTPs. A homogeneous phase detectionmethod based on fluorescence resonance energy transfer has beendescribed by Chen and Kwok (1997) and, Chen and Kwok (Nucleic AcidsResearch 25:347-353 1997) and Chen et al. (Proc. Natl. Acad. Sci. USA94/20 10756-10761,1997), the disclosures of which are incorporatedherein by reference in their entireties). In this method, amplifiedgenomic DNA fragments containing polymorphic sites are incubated with a5′-fluorescein-labeled primer in the presence of allelic dye-labeleddideoxyribonucleoside triphosphates and a modified Taq polymerase. Thedye-labeled primer is extended one base by the dye-terminator specificfor the allele present on the template. At the end of the genotypingreaction, the fluorescence intensities of the two dyes in the reactionmixture are analyzed directly without separation or purification. Allthese steps can be performed in the same tube and the fluorescencechanges can be monitored in real time. Alternatively, the extendedprimer may be analyzed by MALDI-TOF Mass Spectrometry. The base at thepolymorphic site is identified by the mass added onto themicrosequencing primer (see Haff and Smirnov, 1997, Genome Research,7:378-388, 1997, the disclosure of which is incorporated herein byreference in its entirety). In another example, Pastinen et al., (GenomeResearch 7:606-614, 1997), the disclosure of which is incorporatedherein by reference in its entirety) describe a method for multiplexdetection of single nucleotide polymorphism in which the solid phaseminisequencing principle is applied to an oligonucleotide array format.High-density arrays of DNA probes attached to a solid support (DNAchips) are further described below.

Other assays include mismatch detection assays, based on the specificityof polymerases and ligases. Polymerization reactions places particularlystringent requirements on correct base pairing of the 3′ end of theamplification primer and the joining of two oligonucleotides hybridizedto a target DNA sequence is quite sensitive to mismatches close to theligation site, especially at the 3′ end.

A preferred method of determining the identity of the nucleotide presentat an allele involves nucleic acid hybridization. Any hybridizationassay may be used including Southern hybridization, Northernhybridization, dot blot hybridization and solid-phase hybridization (seeSambrook et al., Molecular Cloning—A Laboratory Manual, Second Edition,Cold Spring Harbor Press, N.Y., 1989), the disclosure of which isincorporated herein by reference in its entirety). Hybridization refersto the formation of a duplex structure by two single stranded nucleicacids due to complementary base pairing. Hybridization can occur betweenexactly complementary nucleic acid strands or between nucleic acidstrands that contain minor regions of mismatch. Specific probes can bedesigned that hybridize to one form of a biallelic marker and not to theother and therefore are able to discriminate between different allelicforms. Allele-specific probes are often used in pairs, one member of apair showing perfect match to a target sequence containing the originalallele and the other showing a perfect match to the target sequencecontaining the alternative allele. Hybridization conditions should besufficiently stringent that there is a significant difference inhybridization intensity between alleles, and preferably an essentiallybinary response, whereby a probe hybridizes to only one of the alleles.Stringent, sequence specific hybridization conditions, under which aprobe will hybridize only to the exactly complementary target sequenceare well known in the art (Sambrook et al., 1989). The detection ofhybrid duplexes can be carried out by a number of methods. Variousdetection assay formats are well known which utilize detectable labelsbound to either the target or the probe to enable detection of thehybrid duplexes. Typically, hybridization duplexes are separated fromunhybridized nucleic acids and the labels bound to the duplexes are thendetected. Further, standard heterogeneous assay formats are suitable fordetecting the hybrids using the labels present on the primers andprobes. (see Landegren U. et al., Genome Research, 8:769-776,1998, thedisclosure of which is incorporated herein by reference in itsentirety).

Hybridization assays based on oligonucleotide arrays rely on thedifferences in hybridization stability of short oligonucleotides toperfectly matched and mismatched target sequence variants. Efficientaccess to polymorphism information is obtained through a basic structurecomprising high-density arrays of oligonucleotide probes attached to asolid support (e.g., the chip) at selected positions. Chips of variousformats for use in detecting biallelic polymorphisms can be produced ona customized basis by Affymetrix (GeneChip), Hyseq (HyChip andHyGnostics), and Protogene Laboratories.

In general, these methods employ arrays of oligonucleotide probes thatare complementary to target nucleic acid sequence segments from anindividual which, target sequences include a polymorphic marker. EP785280, the disclosure of which is incorporated herein by reference inits entirety, describes a tiling strategy for the detection of singlenucleotide polymorphisms. Briefly, arrays may generally be “tiled” for alarge number of specific polymorphisms, further described in PCTapplication No. WO 95/11995, the disclosure of which is incorporatedherein by reference in its entirety. Upon completion of hybridizationwith the target sequence and washing of the array, the array is scannedto determine the position on the array to which the target sequencehybridizes. The hybridization data from the scanned array is thenanalyzed to identify which allele or alleles of the biallelic marker arepresent in the sample. Hybridization and scanning may be carried out asdescribed in PCT application No. WO 92/10092 and WO 95/11995 and U.S.Pat. No. 5,424,186, the disclosures of which are incorporated herein byreference in their entireties. Solid supports and polynucleotides of thepresent invention attached to solid supports are further described in“Oligonucleotide Probes And Primers”.

Detecting Chemokines

Some aspects of the present invention relate to the detection ofchemokines by contacting a chemokine or a sample containing a chemokinewith a THAP-type chemokine-binding agent. In some embodiments, thechemokines or the THAP-type chemokine-binding agents are labeled. Manylabels and methods of conjugating such labels to a chemokine or aTHAP-type chemokine-binding agent are known in the art. Additionally,labeled molecules, such as antibodies, which have an affinity for aTHAP-type chemokine-binding agent can be used to detect the chemokinethat is bound to a THAP-type chemokine-binding agent using a number ofassay formats that are well known in the art.

An exemplary method for detecting the presence (quantitative or not) orabsence of a chemokine, including, but not limited to, a chemokine in abiological sample, involves obtaining a chemokine or a sample containinga chemokine and contacting it with a compound or an agent capable ofdetecting the chemokine. In some embodiments, such an agent is aTHAP-type chemokine-binding agent. Chemokines which can be detectedusing a method that employs a THAP-type chemokine-binding agent include,but are not limited to, XCL1, XCL2, CCL1, CCL2, CCL3, CCL3L1, SCYA3L2,CCL4, CCL4L, CCL5, CCL6, CCL7, CCL8, SCYA9, SCYA10, CCL11, SCYA12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22,CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, clone 391, CARP CC-1, CCL1,CK-1, regakine-1, K203, CXCL1, CXCL1P, CXCL2, CXCL3, PF4, PF4V1, CXCL5,CXCL6, PPBP, SPBPBP, IL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14, CXCL15,CXCL16, NAP-4, LFCA-1, Scyba, JSC, VHSV-induced protein, CX3CL1 andfCL1.

In some embodiments, the detection method comprises detecting, in abiological sample, such as a tissue or fluid sample from a subject (suchas, a human patient), the presence or absence of a chemokine bycontacting the biological sample with a THAP-type chemokine-bindingagent and detecting a complex between the chemokine and the THAP-typechemokine-binding agent or detecting a THAP-type chemokine-binding agentwhich was previously bound to the chemokine but which has been releasedfrom the chemokine.

In some embodiments of the present invention, the THAP-typechemokine-binding agent is labeled directly. In other embodiments, theTHAP-type chemokine-binding agent is detected using a labeled antibodyhaving affinity for the THAP-type chemokine-binding agent. Suchantibodies may directly carry the detectable label or be recognized by alabeled second antibody. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)₂) can be used. The term “labeled”, with regard to theantibody or other detectable molecule, is intended to encompass directlabeling of the antibody or molecule by coupling (i.e., physicallylinking) a detectable substance to the antibody or molecule, as well asindirect labeling of the antibody or molecule by reactivity with anotherreagent that is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a THAP-type chemokine-binding agent withbiotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Accordingly, thedetection method can be used to detect a chemokine in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of a chemokine include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Invivo techniques for detection of a chemokine include introducing into asubject a labeled THAP-type chemokine-binding agent. For example, theTHAP-type chemokine-binding agent can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

Other aspects of the present invention relate to a system for chemokinedetection. Such a chemokine detection system comprises a THAP-typechemokine-binding agent bound to a solid support. A number of adequatesolid support materials are known in the art and include, but are notlimited to, cellulose, nylon or other polymer backings, plastics such asmicrotiter plates, synthetic beads and resins such as sepharose, glass,magnetic beads, latex particles, sheep (or other animal) red bloodcells, duracytes and others. Suitable methods for immobilizing theTHAP-type chemokine-binding agent to the solid support are well known inthe art.

Some embodiments of the present invention relate to kits which comprisea THAP-type chemokine-binding agent and instructions which describedetecting or inhibiting chemokines with the THAP-type chemokine-bindingagent. For example, the kit includes an ampule of THAP-typechemokine-binding agent that is stored so as to prevent damage orinactivation of the agent upon prolonged storage. Such methods caninclude, but are not limited to, lyophilization and freezing in anappropriate buffer. The kit also can contain chemokines to serve as apositive control sample when the kit is used for chemokine binding,detection or inhibition.

In some embodiments of the present invention, kits are packagedcontaining a heterogeneous mixture of THAP-type chemokine-bindingagents, wherein each of the agents has a different affinity for one ormore chemokines. Alternatively, some kits comprise a panel of THAP-typechemokine-binding agents, wherein each THAP-type chemokine binding agenthas a different affinity for a particular chemokine. For example, thekit can comprise a panel of three THAP-type chemokine-binding agents,wherein the first agent has a high affinity for SLC but a low affinityfor CXCL9, the second agent has a moderate affinity for both SLC andCXCL9, and the third agent has a low affinity for SLC and a highaffinity for CXCL9. Panels of THAP-type chemokine-binding agents can belarger or small than that exemplified above and the number and types ofchemokines that are detected can be more or less than that exemplifiedabove. Kits containing such panels of THAP-type chemokine-binding agentscan be used to reliably distinguish mixed samples of chemokines.Additionally, such panels can be used to bind or inhibit multipledifferent chemokines in a mixed chemokine sample. Having generallydescribed this invention, a further understanding can be obtained byreference to certain specific examples which are provided herein forpurposes of illustration only, and are not intended to be limitingunless otherwise specified.

EXAMPLES Example 1 Isolation of the THAP1 cDNA in a two-hybrid screenwith chemokine SLC/CCL21

In an effort to define the function of novel HEVEC proteins and thecellular pathways involved, we used different baits to screen atwo-hybrid cDNA library generated from microvascular human HEVendothelial cells (HEVEC). HEVEC were purified from human tonsils byimmunomagnetic selection with monoclonal antibody MECA-79 as previouslydescribed (Girard and Springer (1995) Immunity 2:113-123). The SMART PCRcDNA library Construction Kit (Clontech, Palo Alto, Calif., USA) wasfirst used to generate full-length cDNAs from 1 μg HEVEC total RNA.Oligo-dT-primed HEVEC cDNA were then digested with SfiI anddirectionally cloned into pGAD424-Sfi, a two-hybrid vector generated byinserting a SfiI linker(5′-GAATTCGGCCATTATGGCCTGCAGGATCCGGCCGCCTCGGCCCAGGATCC-3′) (SEQ ID NO:181) between EcoRI and BamHI cloning sites of pGAD424 (Clontech). Theresulting pGAD424-HEVEC cDNA two-hybrid library (mean insert size >1 kb,˜3×10⁶ independant clones) was amplified in E. coli. To identifypotential protein partners of chemokine SLC/6Ckine, screening of thetwo-hybrid HEVEC cDNA library was performed using as bait a cDNAencoding the mature form of human SLC/CCL21 (amino acids 24-134, GenBankAccession No: NP_(—)002980, SEQ ID NO: 182), amplified by PCR from HEVECRNA with primers hSLC.5′ (5′-GCGGGATCCGTAGTGATGGAGGGGCTCAGGACTGTTG-3′)(SEQ ID NO: 183) and hSLC.3′ (5′-GCGGGATCCCTATGGCCCTTTAGGGGTCTGTGACC-3′)(SEQ ID NO: 184), digested with BamHI and inserted into the BamHIcloning site of MATCHMAKER two-hybrid system 2 vector pGBT9 (Clontech).Briefly, pGBT9-SLC was cotransformed with the pGAD424-HEVEC cDNA libraryin yeast strain Y190 (Clontech). 1.5×10⁷ yeast transformants werescreened and positive protein interactions were selected by Hisauxotrophy. The plates were incubated at 30° C. for 5 days. Plasmid DNAwas extracted from positive colonies and used to verify the specificityof the interaction by cotransformation in AH109 with pGBT9-SLC orcontrol baits pGBT9, pGBT9-lamin. Eight independent clones isolated inthis two-hybrid screen were characterized. They were found to correspondto a unique human cDNA encoding a novel human protein of 213 aminoacids, designated THAP1, that exhibits 93% identity with its mouseorthologue (FIG. 1A). The only noticeable motifs in the THAP1 predictedprotein sequence were a short proline-rich domain in the middle part anda consensus nuclear localization sequence (NLS) in the carboxy terminalpart (FIG. 1B). Databases searches with the THAP1 sequence failed toreveal any significant similarity to previously characterized proteinswith the exception of the first 90 amino acids that may define a novelprotein motif associated with apoptosis, hereafter referred to as THAPdomain (see FIG. 1B, FIGS. 9A-9C, and FIG. 10).

Example 2 Northern Blot

To determine the tissue distribution of THAP1 mRNA, we performedNorthern blot analysis of 12 different adult human tissues (FIG. 2).Multiple Human Tissues Northern Blots (CLONTECH) were hydridizedaccording to manufacturer's instructions. The probe was a PCR productcorresponding to the THAP1 ORF, ³²P-labeled with the Prime-a-GeneLabeling System (PROMEGA).A 2.2-kb mRNA band was detected in brain,heart, skeletal muscle, kidney, liver, and placenta. In addition to themajor 2.2 kb band, lower molecular weight bands were detected, that arelikely to correspond to alternative splicing or polyadenylation of theTHAP1 pre-mRNA. The presence of THAP1 mRNAs in many different tissuessuggests that THAP1 has a widespread, although not ubiquitous, tissuedistribution in the human body.

Example 3 Analysis of the Subcellular THAP1 Localization

To analyze the subcellular localization of the THAP1 protein, the THAP1cDNA was fused to the coding sequence of GFP (Green FluorescentProtein). The full-length coding region of THAP1 was amplified by PCRfrom HEVEC cDNA with primers 2HMR10(5′-CCGAATTCAGGATGGTGCAGTCCTGCTCCGCCT-3′) (SEQ ID NO: 185) and 2HMR9(5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQ ID NO: 186),digested with EcoRI and BamHI, and cloned in frame downstream of theEnhanced Green Fluorescent Protein (EGFP) ORF in pEGFP.C2 vector(Clontech) to generate pEGFP.C2-THAP1. The GFP/THAP1 expressionconstruct was then transfected into human primary endothelial cells fromumbilical vein (HUVEC, PromoCell, Heidelberg, Germany). HUVEC were grownin complete ECGM medium (PromoCell, Heidelberg, Germany), plated oncoverslips and transiently transfected in RPMI medium using GeneJammertransfection reagent according to manufacturer instructions (Stratagene,La Jolla, Calif., USA). Analysis by fluorescence microscopy 24 h laterrevealed that the GFP/THAP1 fusion protein localizes exclusively in thenucleus with both a diffuse distribution and an accumulation intospeckles while GFP alone exhibits only a diffuse staining over theentire cell. To investigate the identity of the speckled domains withwhich GFP/THAP1 associates, we used indirect immunofluorescencemicroscopy to examine a possible colocalization of the nuclear dotscontaining GFP/THAP1 with known nuclear domains (replication factories,splicing centers, nuclear bodies).

Cells transfected with GFP-tagged expression constructs were allowed togrow for 24 h to 48 h on coverslips. Cells were washed twice with PBS,fixed for 15 min at room temperature in PBS containing 3.7%formaldehyde, and washed again with PBS prior to neutralization with 50mM NH₄Cl in PBS for 5 min at room temperature. Following one more PBSwash, cells were permeabilized 5 min at room temperature in PBScontaining 0.1% Triton-X100, and washed again with PBS. Permeabilizedcells were then blocked with PBS-BSA (PBS with 1% bovine serum albumin)for 10′ and then incubated 2 hr at room temperature with the followingprimary antibodies diluted in PBS-BSA: rabbit polyclonal antibodiesagainst human Daxx ( 1/50, M-112, Santa Cruz Biotechnology) or mousemonoclonal antibodies anti-PML (mouse IgG1, 1/30, mAb PG-M3 from Dako,Glostrup, Denmark). Cells were then washed three times 5 min at roomtemperature in PBS-BSA, and incubated for 1 hr with Cy3 (redfluorescence)-conjugated goat anti-mouse or anti-rabbit IgG ( 1/1000,Amersham Pharmacia Biotech) secondary antibodies, diluted in PBS-BSA.After extensive washing in PBS, samples were air dried and mounted inMowiol. Images were collected on a Leica confocal laser scanningmicroscope. The GFP (green) and Cy3 (red) fluorescence signals wererecorded sequentially for identical image fields to avoid cross-talkbetween the channels.

This analysis revealed that GFP-THAP1 staining exhibits a completeoverlap with the staining pattern obtained with antibodies directedagainst PML. The colocalization of GFP/THAP1 and PML was observed bothin nuclei with few PML-NBs (less than ten) and in nuclei with a largenumber of PML-NBs. Indirect immunofluorescence staining with antibodiesdirected against Daxx, another well characterized component of PML-NBs,was performed to confirm the association of GFP/THAP1 with PML-NBs. Wefound a complete colocalization of GFP/THAP1 and Daxx in PML-NBs.Together, these results reveal that THAP1 is a novel protein associatedwith PML-NBs.

Example 4 Identification of Proteins Interacting with THAP1 in HumanHEVECs: Two-Hybrid Assay

THAP1 Forms a Complex with the Pro-Apoptotic Protein PAR4

To identify potential protein partners of THAP1, screening of thetwo-hybrid HEVEC cDNA library was performed using as a bait the humanTHAP1 full length cDNA inserted into the MATCHMAKER two-hybrid system 3vector pGBKT7 (Clontech). Briefly, the full-length coding region ofTHAP1 was amplified by PCR from HEVEC cDNA with primers 2HMR10(5′-CCGAATTCAGGATGGTGCAGTCCTGCTCCGCCT-3′) (SEQ ID NO: 187) and 2HMR9(5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQ ID NO: 188),digested with EcoRI and BamHI, and cloned in frame downstream of theGal4 Binding Domain (Gal4-BD) in pGBKT7 vector to generate pGBKT7-THAP1.pGBKT7-THAP1 was then cotransformed with the pGAD424-HEVEC cDNA libraryin yeast strain AH109 (Clontech). 1.5×10⁷ yeast transformants werescreened and positive protein interactions were selected by His and Adedouble auxotrophy according to manufacturer's instructions (MATCHMAKERtwo-hybrid system 3, Clontech). The plates were incubated at 30° C. for5 days. Plasmid DNA was extracted from these positive colonies and usedto verify the specificity of the interaction by cotransformation inAH109 with pGBKT7-THAP1 or control baits pGBKT7, pGBKT7-lamin andpGBKT7-hevin. Three clones which specifically interacted with THAP1 wereobtained in the screen; sequencing of these clones revealed threeidentical library plasmids that corresponded to a partial cDNA codingfor the last 147 amino acids (positions 193-342) of the humanpro-apoptotic protein PAR4 (FIG. 3A). Positive interaction between THAP1and Par4 was confirmed using full length Par4 bait (pGBKT-Par4) and prey(pGADT7-Par4). Full-length human Par4 was amplified by PCR from humanthymus cDNA (Clontech), with primers Par4.8(5′-GCGGAATTCATGGCGACCGGTGGCTACCGGACC-3′) (SEQ ID NO: 189) and Par4.5(5′-GCGGGATCCCTCTACCTGGTCAGCTGACCCACAAC-3′) (SEQ ID NO: 190), digestedwith EcoRI and BamHI, and cloned in pGBKT7 and pGADT7 vectors, togenerate pGBKT7-Par4 and pGADT7-Par4. Positive interaction between THAP1and Par4 was confirmed by cotransformation of AH109 with pGBKT7-THAP1and pGADT7-Par4 or pGBKT7-Par4 and pGADT7-THAP1 and selection oftransformants by His and Ade double auxotrophy according tomanufacturer's instructions (MATCHMAKER two-hybrid system 3, Clontech).To generate pGADT7-THAP1, the full-length coding region of THAP1 wasamplified by PCR from HEVEC cDNA with primers 2HMR10(5′-CCGAATTCAGGATGGTGCAGTCCTGCTCCGCCT-3′) (SEQ ID NO: 191) and 2HMR9(5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQ ID NO: 192),digested with EcoRI and BamHI, and cloned in frame downstream of theGal-4 Activation Domain (Gal4-AD) in pGADT7 two-hybrid vector(Clontech).

We then examined whether the leucine zipper/death domain at theC-terminus of Par4, previously shown to be involved in Par4 binding toWT-1 and aPKC, was required for the interaction between THAP1 and Par4.Two Par4 mutants were constructed for that purpose, Par4Δ and Par4DD.Par4Δ lacks the leucine zipper/death domain while Par4DD contains thisdomain. pGBKT7-Par4Δ(amino acids 1-276) and pGADT7-Par4Δ. wereconstructed by sub-cloning a EcoRI-BglII fragment from pGADT7-Par4 intothe EcoRI and BamHI sites of pGBKT7 and pGADT7. Par4DD (amino acids250-342) was amplified by PCR, using pGBKT7-Par4 as template, withprimers Par4. 4 (5′-CGCGAATTCGCCATCATGGGGTTCCCTAGATATAACAGGGATGCAA-3′)(SEQ ID NO: 193) and Par4.5, and cloned into the EcoRI and BamHI sitesof pGBKT7 and pGADT7 to obtain pGBKT7-Par4DD and pGADT7-Par4DD.Two-hybrid interaction between THAP1 and Par4 mutants was tested bycotransformation of AH109 with pGBKT7-THAP1 and pGADT7-Par4Δ orpGADT7-Par4DD and selection of transformants by His and Ade doubleauxotrophy according to manufacturer's instructions (MATCHMAKERtwo-hybrid system 3, Clontech). We found that the Par4 leucinezipper/death domain (Par4DD) is not only required but also sufficientfor the interaction with THAP1 (FIG. 3A). Similar results were obtainedwhen two-hybrid experiments were performed in the opposite orientationusing Par4 or Par4 mutants (Par4Δ and Par4DD) as baits instead of THAP1(FIG. 3A).

Example 5 In vitro THAP1/Par4 Interaction Assay

To confirm the interaction observed in yeast, we performed in vitro GSTpull down assays. Par4DD, expressed as a GST-tagged fusion protein andimmobilized on glutathione sepharose, was incubated with radiolabeled invitro translated THAP1. To generate the GST-Par4DD expression vector,Par4DD (amino acids 250-342) was amplified by PCR with primers Par4.10(5′-GCCGGATCCGGGTTCCCTAGATATAACAGGGATGCAA-3′) (SEQ ID NO: 194) andPar4.5, and cloned in frame downstream of the Glutathion S-TransferaseORF, into the BamHI site of the pGEX-2T prokaryotic expression vector(Amersham Pharmacia Biotech, Saclay, France). GST-Par4DD(amino acids250-342) fusion protein encoded by plasmid pGEX-2T-Par4DD and controlGST protein encoded by plasmid pGEX-2T, were then expressed in E. coliDH5α and purified by affinity chromatography with glutathione sepharoseaccording to supplier's instructions (Amersham Pharmacia Biotech). Theyield of proteins used in GST pull-down assays was determined bySDS-Polyarylamide Gel Electrophoresis (PAGE) and Coomassie blue staininganalysis. In vitro-translated THAP1 was generated with the TNT-coupledreticulocyte lysate system (Promega, Madison, Wis., USA) usingpGBKT7-THAP1 vector as template. 25 μl of ³⁵S-labeled wild-type THAP1was incubated with immobilized GST-Par4 or GST proteins overnight at 4°C., in the following binding buffer: 10 mM NaPO4 pH 8.0, 140 mM NaCl, 3mM MgCl2, 1 mM dithiothreitol (DTT), 0.05% NP40, and 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM Na vanadate, 50 mM, Glycerophosphate, 25μg/ml chymotrypsine, 5 μg/ml aprotinin, and 10 μg/ml leupeptin. Beadswere then washed 5 times in 1 ml binding buffer. Bound proteins wereeluted with 2× Laemmli SDS-PAGE sample buffer, fractionated by 10%SDS-PAGE and visualized by fluorography using Amplify (AmershamPharmacia Biotech). As expected, GST/Par4DD interacted with THAP1 (FIG.3B). In contrast, THAP1 failed to interact with GST beads.

Example 6 In vivo THAP1/Par4 Interaction Assay

To provide further evidence for a physiological interaction betweenTHAP1 and Par4 in vivo interactions between THAP1 and PAR4 wereinvestigated. For that purpose, confocal immunofluorescence microscopywas used to analyze the subcellular localization of epitope-taggedPar4DD in primary human endothelial cells transiently cotransfected withpEF-mycPar4DD eukaryotic expression vector and GFP or GFP-THAP1expression vectors (pEGFP.C2 and pEGFP.C2-THAP1, respectively). Togenerate pEF-mycPar4DD, mycPar4DD (amino acids 250-342) was amplified byPCR using pGBKT7-Par4DD as template, with primers myc.BD7(5′-GCGCTCTAGAGCCATCATGGAGGAGCAGAAGCTGATC-3′) (SEQ ID NO: 195) andPar4.9 (5′-CTTGCGGCCGCCTCTACCTGGTCAGCTGACCCACAAC-3′) (SEQ ID NO: 196),and cloned into the XbaI and NotI sites of the pEF-BOS expression vector(Mizushima and Nagata, Nucleic Acids Research, 18:5322, 1990). Primaryhuman endothelial cells from umbilical vein (HUVEC, PromoCell,Heidelberg, Germany) were grown in complete ECGM medium (PromoCell,Heidelberg, Germany), plated on coverslips and transiently transfectedin RPMI medium using GeneJammer transfection reagent according tomanufacturer instructions (Stratagene, La Jolla, Calif., USA). Cellsco-transfected with pEF-mycPar4DD and GFP-tagged expression constructswere allowed to grow for 24 h to 48 h on coverslips. Cells were washedtwice with PBS, fixed for 15 min at room temperature in PBS containing3.7% formaldehyde, and washed again with PBS prior to neutralizationwith 50 mM NH₄Cl in PBS for 5 min at room temperature. Following onemore PBS wash, cells were permeabilized 5 min at room temperature in PBScontaining 0.1% Triton-X100, and washed again with PBS. Permeabilizedcells were then blocked with PBS-BSA (PBS with 1% bovine serum albumin)for 10′ and then incubated 2 hr at room temperature with mousemonoclonal antibody anti-myc epitope (mouse IgG1, 1/200, Clontech)diluted in PBS-BSA. Cells were then washed three times 5 min at roomtemperature in PBS-BSA, and incubated for 1 hr with Cy3 (redfluorescence)-conjugated goat anti-mouse ( 1/1000, Amersham PharmaciaBiotech) secondary antibodies, diluted in PBS-BSA. After extensivewashing in PBS, samples were air dried and mounted in Mowiol. Imageswere collected on a Leica confocal laser scanning microscope. The GFP(green) and Cy3 (red) fluorescence signals were recorded sequentiallyfor identical image fields to avoid cross-talk between the channels.

In cells transiently co-transfected with pEF-mycPar4DD and GFPexpression vector, ectopically expressed myc-Par4DD was found toaccumulate both in the cytoplasm and the nucleus of the majority of thecells. In contrast, transient cotransfection of pEF-mycPar4DD andGFP-THAP1 expression vectors dramatically shifted myc-Par4DD from adiffuse cytosolic and nuclear localization to a preferential associationwith PML-NBs. The effect of GFP-THAP1 on myc-Par4DD localization wasspecific since it was not observed with GFP-APS kinase-1 (APSK-1), anuclear enzyme unrelated to THAP1 and apoptosis [Besset et al., Faseb J,14:345-354, 2000]. This later result shows that GFP-THAP1 recruitsmyc-Par4DD at PML-NBs and provides in vivo evidence for a directinteraction of THAP1 with the pro-apoptotic protein Par4.

Example 7 Identification of a Novel Arginine-Rich Par4 Binding Motif

To identify the sequences mediating THAP1 binding to Par4, a series ofTHAP 1 deletion constructs was generated. Both amino-terminal (THAP1-C1,—C2, —C3) and carboxy-terminal (THAP1-N1, —N2, —N3) deletion mutants(FIG. 4A) were amplified by PCR using plasmid pEGFP.C2-THAP1 as atemplate and the following primers: 2HMR12(5′-GCGGAATTCAAAGAAGATCTTCTGGAGCCACAGGAAC-3′) (SEQ ID NO: 197) and 2HMR9(5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQ ID NO: 198) forTHAP1-C1 (amino acids 90-213); PAPM2(5′-GCGGAATTCATGCCGCCTCTTCAGACCCCTGTTAA-3′) (SEQ ID NO: 199) and 2HMR9for THAP1-C2 (amino acids 120-213); PAPM3(5′-GCGGAATTCATGCACCAGCGGAAAAGGATTCATCAG-3′) (SEQ ID NO: 200) and 2HMR9for THAP1-C3 (amino acids 143-213); 2HMR10(5′-CCGAATTCAGGATGGTGCAGTCCTGCTCCGCCT-3′) (SEQ ID NO: 201) and 2HMR17(5′-GCGGGATCCCTTGTCATGTGGCTCAGTACAAAGAAATAT-3′) (SEQ ID NO: 202) forTHAP1-N1 (amino acids 1-90); 2HMR10 and PAPN2(5′-CGGGATCCTGTGCGGTCTTGAGCTTCTTTCTGAG-3′) (SEQ ID NO: 203) for THAP1-N2(amino acids 1-166); and 2HMR10 and PAPN3(5′-GCGGGATCCGTCGTCTTTCTCTTTCTGGAAGTGAAC-3′) (SEQ ID NO: 204) forTHAP1-N3 (amino acids 1-192).

The PCR fragments, thus obtained, were digested with EcoRI and BamHI,and cloned in frame downstream of the Gal4 Binding Domain (Gal4-BD) inpGBKT7 two-hybrid vector (Clontech) to generate pGBKT7-THAP1-C1, —C2,—C3, —N1, —N2 or —N3, or downstream of the Enhanced Green FluorescentProtein (EGFP) ORF in pEGFP.C2 vector (Clontech) to generatepEGFP.C2-THAP1-C1, —C2, —C3, —N1, —N2 or —N3.

Two-hybrid interaction between THAP1 mutants and Par4DD was tested bycotransformation of AH109 with pGBKT7-THAP1-C1, —C2, —C3, —N1, —N2 or—N3 and pGADT7-Par4DD and selection of transformants by His and Adedouble auxotrophy according to manufacturer's instructions (MATCHMAKERtwo-hybrid system 3, Clontech). Positive two-hybrid interaction withPar4DD was observed with mutants THAP1-C1, —C2, —C3, -and —N3 but notwith mutants THAP1-N1 and —N2, suggesting the Par4 binding site is foundbetween THAP1 residues 143 and 192.

THAP1 mutants were also tested in the in vitro THAP1/Par4 interactionassay. In vitro-translated THAP1 mutants were generated with theTNT-coupled reticulocyte lysate system (Promega, Madison, Wis., USA)using pGBKT7-THAP1-C1, —C2, —C3, —N1, —N2 or —N3 vector as template. 25μl of each ³⁵S-labelled THAP1 mutant was incubated with immobilized GSTor GST-Par4 protein overnight at 4° C., in the following binding buffer:10 mM NaPO4 pH 8.0, 140 mM NaCl, 3 mM MgCl2, 1 mM dithiothreitol (DTT),0.05% NP40, and 0.2 mM phenylmethyl sulphonyl fluoride (PMSF), 1 mM NaVanadate, 50 mM β Glycerophosphate, 25 μg/ml chimotrypsine, 5 μg/mlaprotinin, 10 μg/ml Leupeptin. Beads were then washed 5 times in 1 mlbinding buffer. Bound proteins were eluted with 2× Laemmli SDS-PAGEsample buffer, fractionated by 10% SDS-PAGE and visualized byfluorography using Amplify (Amersham Pharmacia Biotech). As expected,THAP1-C1, —C2, —C3, -and —N3 interacted with GST/Par4DD (FIG. 4B). Incontrast, THAP1-N1 and —N2 failed to interact with GST/Par4DD beads.

Finally, Par4 binding activity of THAP1 mutants was also analyzed by thein vivo THAP1/Par4 interaction assay as described in Example 6 usingpEF-mycPar4DD and pEGFP.C2-THAP1-C1, —C2, —C3, —N1, —N2 or —N3expression vectors.

Essentially identical results were obtained with the three THAP1/Par4interactions assays (FIG. 4A). That is, the Par4 binding site was foundbetween residues 143 and 192 of human THAP1. Comparison of this regionwith the Par4 binding domain of mouse ZIP kinase, anotherPar4-interacting protein, revealed the existence of a conserved argininerich-sequence motif (SEQ ID NOs: 205, 263 and 15), that may correspondto the Par4 binding site (FIG. 5A). Mutations in this argininerich-sequence motif were generated by site directed mutagenesis. Thesetwo novel THAP1 mutants, THAP1 RR/AA (replacement of residues R171A andR172A) and THAP1ΔQRCRR (deletion of residues 168-172), were generated bytwo successive rounds of PCR using pEGFP.C2-THAP1 as template andprimers 2HMR10 and 2HMR9 together with primers RR/AA-1(5′-CCGCACAGCAGCGATGCGCTGCTCAAGAACGGCAGCTTG-3′) (SEQ ID NO: 206) andRR/AA-2 (5′-CAAGCTGCCGTTCTTGAGCAGCGCATCGCTGCTGTGCGG-3′) (SEQ ID NO: 207)for mutant THAP1 RR/AA or primers ΔRR-1(5′-GCTCAAGACCGCACAGCAAGAACGGCAGCTTG-3′(SEQ ID NO: 208) and ΔRR-2(5′-CAAGCTGCCGTTCTTGCTGTGCGGTCTTGAGC-3═) (SEQ ID NO: 209) for mutantTHAP1ΔQRCRR. The resulting PCR fragments were digested with EcoRI andBamHI, and cloned in frame downstream of the Gal4 Binding Domain(Gal4-BD) in pGBKT7 two-hybrid vector (Clontech) to generatepGBKT7-THAP1-RR/AA and -Δ(QRCRR), or downstream of the Enhanced GreenFluorescent Protein (EGFP) ORF in pEGFP.C2 vector (Clontech) to generatepEGFP.C2-THAP1-RR/AA and -Δ(QRCRR). THAP1 RR/AA and THAP1ΔQRCRR THAP1mutants were then tested in the three THAP1/Par4 interaction assays(two-hybrid assay, in vitro THAP1/Par4 interaction assay, in vivoTHAP1/Par4 interaction assay) as described above for the THAP1-C1, —C2,—C3, —N1, —N2 or —N3 mutants. This analysis revealed that the twomutants were deficient for interaction with Par4 in all three assays(FIG. 5B), indicating that the novel arginine-rich sequence motif, wehave identified, is a novel Par4 binding motif.

Example 8 PAR4 is a Novel Component of PML-NBs that Colocalizes withTHAP1 in vivo

We then wished to determine if PAR4 colocalizes with THAP1 in vivo inorder to provide further evidence for a physiological interactionbetween THAP1 and PAR4. We first analyzed Par4 subcellular localizationin primary human endothelial cells. Confocal immunofluorescencemicroscopy using affinity-purified anti-PAR4 antibodies (Sells et al.,1997; Guo et al; 1998) was performed on HUVEC endothelial cells fixedwith methanol/acetone, which makes PML-NBs components accessible forantibodies (Sternsdorf et al., 1997). Cells were fixed in methanol for 5min at −20° C., followed by incubation in cold acetone at −20° C. for 30sec. Permeabilized cells were then blocked with PBS-BSA (PBS with 1%bovine serum albumin) for 10′ and then incubated 2 hr at roomtemperature with rabbit polyclonal antibodies against human Par4 ( 1/50,R-334, Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and mousemonoclonal antibody anti-PML (mouse IgG1; 1/30, mAb PG-M3 from Dako,Glostrup, Denmark). Cells were then washed three times 5 min at roomtemperature in PBS-BSA, and incubated for 1 hr with Cy3 (redfluorescence)-conjugated goat anti-rabbit IgG ( 1/1000, AmershamPharmacia Biotech) and FITC-labeled goat anti-mouse-IgG ( 1/40, ZymedLaboratories Inc., San Francisco, Calif., USA) secondary antibodies,diluted in PBS-BSA. After extensive washing in PBS, samples were airdried and mounted in Mowiol. Images were collected on a Leica confocallaser scanning microscope. The FITC (green) and Cy3 (red) fluorescencesignals were recorded sequentially for identical image fields to avoidcross-talk between the channels. This analysis showed an association ofPAR4 immunoreactivity with nuclear dot-like structures, in addition todiff-use nucleoplasmic and cytoplasmic staining. Double immunostainingwith anti-PML antibodies, revealed that the PAR4 foci colocalizeperfectly with PML-NBs in cell nuclei. Colocalization of Par4 withGFP-THAP1 in PML-NBs was analyzed in transfected HUVEC cells expressingectopic GFP-THAP1. HUVEC were grown in complete ECGM medium (PromoCell,Heidelberg, Germany), plated on coverslips and transiently transfectedwith GFP/THAP1 expression construct (pEGFP.C2-THAP1) in RPMI mediumusing GeneJammer transfection reagent according to manufacturerinstructions (Stratagene, La Jolla, Calif., USA). Analysis oftransfected cells by indirect immunofluorescence microscopy 24 h later,with anti-Par4 rabbit antibodies, revealed that all endogenous PAR4 focicolocalize with ectopic GFP-THAP1 in PML-NBs further confirming theassociation of the THAP1/PAR4 complex with PML-NBs in vivo.

Example 9 PML recruits the THAP1/PAR4 complex to PML-NBs

Since it has been shown that PML plays a critical role in the assemblyof PML-NBs by recruiting other components, we next wanted to determinewhether PML plays a role in the recruitment of the THAP1/PAR4 complex toPML-NBs. For this purpose, we made use of the observation that bothendogenous PAR4 and ectopic GFP-THAP1 do not accumulate in PML-NBs inhuman Hela cells. Expression vectors for GFP-THAP1 and HA-PML (orHA-SP100) were cotransfected into these cells and the localization ofendogenous PAR4, GFP-THAP1 and HA-PML (or HA-SP100) was analyzed bytriple staining confocal microscopy.

Human Hela cells (ATCC) were grown in Dulbecco's Modified Eagle's Mediumsupplemented with 10% Fetal Calf Serurn and 1% Penicillin-streptomycin(all from Life Technologies, Grand Island, N.Y., USA), plated oncoverslips, and transiently transfected with calcium phosphate methodusing 2 μg pEGFP.C2-THAP1 and pcDNA.3-HA-PML3 or pSG5-HA-Sp100 (a giftfrom Dr Dejean, Institut Pasteur, Paris, France) plasmid DNA.pcDNA.3-HA-PML3 was constructed by sub-cloning a BglII-BamHI fragmentfrom pGADT7-HA-PML3 into the BamHI site of pcDNA3 expression vector(Invitrogen, San Diego, Calif., USA). To generate pGADT7-HA-PML3, PML3ORF was amplified by PCR, using pACT2-PML3 (a gift from Dr De Thé,Paris, France) as template, with primers PML-1(5′-GCGGGATCCCTAAATTAGAAAGGGGTGGGGGTAGCC-3′) (SEQ ID NO: 210) and PML-2(5′-GCGGAATTCATGGAGCCTGCACCCGCCCGATC-3′) (SEQ ID NO: 211), and clonedinto the EcoRI and BamHI sites of pGADT7.

Hela cells transfected with GFP-tagged and HA-tagged expressionconstructs were allowed to grow for 24 h to 48 h on coverslips. Cellswere washed twice with PBS, fixed in methanol for 5 min at −20° C.,followed by incubation in cold acetone at −20° C. for 30 sec.Permeabilized cells were then blocked with PBS-BSA (PBS with 1% bovineserum albumin) for 10′ and then incubated 2 hr at room temperature withthe following primary antibodies diluted in PBS-BSA: rabbit polyclonalantibodies against human Par4 ( 1/50, R-334, Santa Cruz Biotechnology,Santa Cruz, Calif., USA) and mouse monoclonal antibody anti-HA tag(mouse IgG1, 1/1000, mAb 16B12 from BabCO, Richmond, Calif., USA). Cellswere then washed three times 5 min at room temperature in PBS-BSA, andincubated for 1 hr with Cy3 (red fluorescence)-conjugated goatanti-rabbit IgG ( 1/1000, Amersham Pharmacia Biotech) and AlexaFluor-633 (blue fluorescence) goat anti-mouse IgG conjugate ( 1/100,Molecular Probes, Eugene, Oreg., USA) secondary antibodies, diluted inPBS-BSA. After extensive washing in PBS, samples were air dried andmounted in Mowiol. Images were collected on a Leica confocal laserscanning microscope. The GFP (green), Cy3 (red) and Alexa 633 (blue)fluorescence signals were recorded sequentially for identical imagefields to avoid cross-talk between the channels.

In Hela cells transfected with HA-PML, endogenous PAR4 and GFP-THAP1were recruited to PML-NBs, whereas in cells transfected with HA-SP100,both PAR4 and GFP-THAP1 exhibited diffuse staining without accumulationin PML-NBs. These findings indicate that recruitment of the THAP1/PAR4complex to PML-NBs depends on PML but not SP100.

Example 10 THAP 1 is an Apoptosis Inducing Polypeptide

THAP is a Novel Proapoptotic Factor

Since PML and PML-NBs have been linked to regulation of cell death andPAR4 is a well established pro-apoptotic factor, we examined whetherTHAP1 can modulate cell survival. Mouse 3T3 cells, which have previouslybeen used to analyze the pro-apoptotic activity of PAR4 (Diaz-Meco etal, 1996; Berra et al., 1997), were transfected with expression vectorsfor GFP-THAP1, GFP-PAR4 and as a negative control GFP-APS kinase-1(APSK-1), a nuclear enzyme unrelated to THAP1 and apoptosis (Girard etal., 1998; Besset et al., 2000). We then determined whether ectopicexpression of THAP1 enhances the apoptotic response to serum withdrawal.Transfected cells were deprived of serum for up to twenty four hours andcells with apoptotic nuclei, as revealed by DAPI staining and in situTUNEL assay, were counted.

Cell death assays: Mouse 3T3-TO fibroblasts were seeded on coverslips in12-well plates at 40 to 50% confluency and transiently transfected withGFP or GFP-fusion protein expression vectors using Lipofectamine Plusreagent (Life Technologies) according to supplier's instructions. After6 h at 37° C., the DNA-lipid mixture was removed and the cells wereallowed to recover in complete medium for 24 h. Serum starvation oftransiently transfected cells was induced by changing the medium to 0%serum, and the amount of GFP-positive apoptotic cells was assessed 24 hafter induction of serum starvation. Cells were fixed in PBS containing3.7% formaldehyde and permeabilized with 0.1% Triton-X100 as describedunder immunofluorescence, and apoptosis was scored by in situ TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)and/or DAPI (4,6-Diamidino-2-phenylindole) staining of apoptotic nucleiexhibiting nuclear condensation. The TUNEL reaction was performed for 1hr at 37° C. using the in situ cell death detection kit, TMR red (RocheDiagnostics, Meylan, France). DAPI staining with a final concentrationof 0.2 μg/ml was performed for 10 min at room temperature. At least 100cells were scored for each experimental point using a fluorescencemicroscope.

Basal levels of apoptosis in the presence of serum ranged from 1-3%.Twenty four hours after serum withdrawal, apoptosis was found in 18% ofuntransfected 3T3 cells and in 3T3 cells overexpressingGFP-APSK-1.Levels of serum withdrawal induced apoptosis weresignificantly increased to about 70% and 65% in cells overexpressingGFP-PAR4 and GFP-THAP1, respectively (FIG. 6A). These resultsdemonstrate that THAP1, similarly to PAR4, is an apoptosis inducingpolypeptide.

TNFα-induced apoptosis assays were performed by incubating transientlytransfected cells in complete medium containing 30 ng/ml of mTNFα (R &D, Minneapolis, Minn., USA) for 24 h. Apoptosis was scored as describedfor serum withdrawal-induced apoptosis. The results are shown in FIG.6B. As shown in FIG. 6B, THAP1 induced apoptosis.

Example 11 The THAP Domain is Essential for THAP1 Pro-Apoptotic Activity

To determine the role of the amino-terminal THAP domain (amino acids 1to 89) in the functional activity of THAP1, we generated a THAP1 mutantthat is deleted of the THAP domain (THAP1ΔTHAP). THAP1ΔTHAP (amino acids90-213) was amplified by PCR, using pEGFP.C2-THAP1 as template, withprimers 2HMR]2 (5′-GCGGAATTCAAAGAAGATCTTCTGGAGCCACAGGAAC-3′) (SEQ ID NO:212) and 2HMR9 (5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQ IDNO: 213), digested with EcoRI and BamHI, and cloned in pGBKT7 andpEGFP-C2 vectors, to generate pGBKT7-TRAP1ΔTHAP and pEGFP.C2-THAP1ΔTHAPexpression vectors. The role of the THAP domain in PML NBs localization,binding to Par4, or pro-apoptotic activity of THAP1 was then analyzed.

To analyze the subcellular localization of THAP1ΔTHAP, theGFP/THAP1ΔTHAP expression construct was transfected into human primaryendothelial cells from umbilical vein (HUVEC, PromoCell, Heidelberg,Germany). HUVEC were grown in complete ECGM medium (PromoCell,Heidelberg, Germany), plated on coverslips and transiently transfectedin RPMI medium using GeneJammer transfection reagent according tomanufacturer instructions (Stratagene, La Jolla, Calif., USA).Transfected cells were allowed to grow for 48 h on coverslips. Cellswere then washed twice with PBS, fixed for 15 min at room temperature inPBS containing 3.7% formaldehyde, and washed again with PBS prior toneutralization with 50mM NH₄Cl in PBS for 5 min at room temperature.Following one more PBS wash, cells were permeabilized 5 min at roomtemperature in PBS containing 0.1% Triton-X100, and washed again withPBS. Permeabilized cells were then blocked with PBS-BSA (PBS with 1%bovine serum albumin) for 10′ and then incubated 2 hr at roomtemperature with mouse monoclonal antibody anti-PML (mouse IgG1, 1/30,mAb PG-M3 from Dako, Glostrup, Denmark) diluted in PBS-BSA. Cells werethen washed three times 5 min at room temperature in PBS-BSA, andincubated for 1 hr with Cy3 (red fluorescence)-conjugated goatanti-mouse IgG ( 1/1000, Amersham Pharmacia Biotech) secondaryantibodies, diluted in PBS-BSA. After extensive washing in PBS, sampleswere air dried and mounted in Mowiol. Images were collected on a Leicaconfocal laser scanning microscope. The GFP (green) and Cy3 (red)fluorescence signals were recorded sequentially for identical imagefields to avoid cross-talk between the channels.

This analysis revealed that GFP-THAP1ΔTHAP staining exhibits a completeoverlap with the staining pattern obtained with antibodies directedagainst PML, indicating the THAP domain is not required for THAP1localization to PML NBs.

To examine the role of the THAP domain in binding to Par4, we performedin vitro GST pull down assays. Par4DD, expressed as a GST-tagged fusionprotein and immobilized on glutathione sepharose, was incubated withradiolabeled in vitro translated THAP1ΔTHAP. In vitro-translatedTHAP1ΔTHAP was generated with the TNT-coupled reticulocyte lysate system(Promega, Madison, Wis., USA) using pGBKT7-THAP1ΔTHAP vector astemplate. 25 μl of ³⁵S-labelled THAP1ΔΔTHAP was incubated withimmobilized GST-Par4 or GST proteins overnight at 4° C., in thefollowing binding buffer: 10 mM NaPO4 pH 8.0, 140 mM NaCl, 3 mM MgCl2, 1mM dithiothreitol (DTT), 0.05% NP40, and 0.2 mM phenylmethyl sulphonylfluoride (PMSF), 1 mM Na Vanadate, 50 mM , β Glycerophosphate, 25 μg/mlchimotrypsine, 5 μg/ml aprotinin, 10 μg/ml Leupeptin. Beads were thenwashed 5 times in 1 ml binding buffer. Bound proteins were eluted with2× Laemmli SDS-PAGE sample buffer, fractionated by 10% SDS-PAGE andvisualized by fluorography using Amplify (Amersham Pharmacia Biotech).

This analysis revealed that THAP1ΔTHAP interacts with GST/Par4DD,indicating that the THAP domain is not involved in THAP1/Par4interaction (FIG. 7A).

To examine the role of the THAP domain in THAP1 pro-apoptotic activity,we performed cell death assays in mouse 3T3 cells. Mouse 3T3-TOfibroblasts were seeded on coverslips in 12-well plates at 40 to 50%confluency and transiently transfected with GFP-APSK1, GFP-THAP1 orGFP-THAP1ΔTHAP fusion proteins expression vectors using LipofectaminePlus reagent (Life Technologies) according to supplier's instructions.After 6 h at 37° C., the DNA-lipid mixture was removed and the cellswere allowed to recover in complete medium for 24 h. Serum starvation oftransiently transfected cells was induced by changing the medium to 0%serum, and the amount of GFP-positive apoptotic cells was assessed 24 hafter induction of serum starvation. Cells were fixed in PBS containing3.7% formaldehyde and permeabilized with 0.1% Triton-X100 as describedunder immunofluorescence, and apoptosis was scored by in situ TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)and/or DAPI (4,6-Diamidino-2-phenylindole) staining of apoptotic nucleiexhibiting nuclear condensation. The TUNEL reaction was performed for 1hr at 37° C. using the in situ cell death detection kit, TMR red (RocheDiagnostics, Meylan, France). DAPI staining with a final concentrationof 0.2 μg/ml was performed for 10 min at room temperature. At least 100cells were scored for each experimental point using a fluorescencemicroscope.

Twenty four hours after serum withdrawal, apoptosis was found in 18% ofuntransfected 3T3 cells and in 3T3 cells overexpressing GFP-APSK-1.Levels of serum withdrawal induced apoptosis were significantlyincreased to about 70% in cells overexpressing GFP-THAP1. Deletion ofthe THAP domain abrogated most of this effect sinceserum-withdrawal-induced apoptosis was reduced to 28% in cellsoverexpressing GFP-THAP1ΔTHAP (FIG. 7B). These results indicate that theTHAP domain, although not required for THAP1 PML-NBs localization andPar4 binding, is essential for THAP1 pro-apoptotic activity.

Example 12 The THAP Domain Defines a Novel Family of Proteins the THAPFamily

To discover novel human proteins homologous to THAP1 and/or containingTHAP domains, GenBank non-redundant, human EST and draft human genomedatabases at the National Center for Biotechnology Information(www.ncbi.nlm.nih.gov) were searched with both the nucleotide and aminoacid sequences of THAP1, using the programs BLASTN, TBLASTN and BLASTP(Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J.(1990). Basic local alignment search tool. J Mol Biol 215: 403-410).This initial step enabled us to identify 12, distinct humanTHAP-containing, proteins (hTHAP0 to hTHAP11; FIG. 8). In the case ofthe partial length sequences, assembly of overlapping ESTs together withGENESCAN (Burge, C.and Karlin, S. (1997). Prediction of complete genestructures in human genomic DNA. J Mol Biol 268: 78-94) and GENEWISE(Jareborg, N., Birney, E. and Durbin, R. (1999). Comparative analysis ofnoncoding regions of 77 orthologous mouse and human gene pairs. GenomeRes 9: 815-824) gene predictions on the corresponding genomic DNAclones, was used to define the full length human THAP proteins as wellas their corresponding cDNAs and genes. CLUSTALW (Higgins, D. G.,Thompson, J. D. and Gibson, T. J. (1996). Using CLUSTAL for multiplesequence alignments. Methods Enzymol 266: 383-402) was used to carry outthe alignment of the 12 human THAP domains with the DNA binding domainof Drosophila P-element transposase (Lee, C. C., Beall, E. L., and Rio,D. C. (1998) Embo J. 17:4166-74), which was colored using the computerprogram Boxshade (www.ch.embnet.org/software/BOX_form.html) (see FIGS.9A and 9B). Equivalent approach to the one described above was used inorder to identify the mouse, rat, pig, and various other orthologs ofthe human THAP proteins (FIG. 9C). Altogether, the in silico andexperimental approaches led to the discovery of 12 distinct humanmembers (hTHAP0 to hTHAP11) of the THAP family of pro-apoptotic factors(FIG. 8).

Example 13 THAP2 and THAP3 Interact with Par-4

To assess whether THAP2 and THAP3 are able to interact with Par-4, yeasttwo hybrid assays using Par-4 wild type bait (FIG. 10B) and in vitro GSTpull down assays (FIG. 10C), were performed as described above (Examples4 and 5). As shown in FIGS. 10B and 10C, THAP2 and THAP3 are able tointeract with Par-4. A sequence alignment showing the comparison of theTHAP domain and the PAR4-binding domain between THAP1, THAP2 and THAP3is shown in FIG. 10A.

Example 14 THAP2 and THAP3 are Able to Induce Apoptosis

Serum-induced or TNFα apoptosis analyses were performed as describedabove (Example 10) in cells transfected with GFP-APSK1, GFP-THAP2 orGFP-THAP3 expression vectors. Apoptosis was quantified by DAPI stainingof apoptotic nuclei 24 hours after serum withdrawal or addition of TNFα.The results are shown in FIG. 11A (serum withdrawal) and FIG. 11B(TNFα). These results indicate that, THAP-2 and THAP3 induce apoptosis.

Example 15 Identification of the SLC/CCL21 Chemokine-Binding Domain ofHuman THAP1

To identify the SLC/CCL21 chemokine-binding domain of human THAP1, aseries of THAP1 deletion constructs was generated as described inExample 7.

Two-hybrid interaction between THAP1 mutants and chemokine SLC/CCL21 wastested by cotransformation of AH109 with pGADT7-THAP1-C1, —C2, —C3, —N1,—N2 or —N3 and pGBKT7-SLC/CCL21 and selection of transformants by Hisand Ade double auxotrophy according to manufacturer's instructions(MATCHMAKER two-hybrid system 3, Clontech). pGBKT7-SLC/CCL21 vector wasgenerated by subcloning the BamHI SLC/CCL21 fragment from pGBT9-SLC (seeexample 1) into the unique BamHI cloning site of vector pGBKT7(Clontech). Positive two-hybrid interaction with chemokine SLC/CCL21 wasobserved with mutants THAP1-C1, —C2, —C3, but not with mutants THAP1-N1,—N2 and —N3, suggesting that the SLC/CCL21 chemokine-binding domain ofhuman THAP1 is found between THAP1 residues 143 and 213 (FIG. 12).

Example 16 In vitro THAP1/Chemokine SLC-CCL21 Interaction Assay

To confirm the interaction observed in yeast two-hybrid system, weperformed in vitro GST pull down assays. THAP1, expressed as aGST-tagged fusion protein and immobilized on glutathione sepharose, wasincubated with radiolabeled in vitro translated SLC/CCL21.

To generate the GST-THAP1 expression vector, the full-length codingregion of THAP1 (amino acids 1-213) was amplified by PCR from HEVEC cDNAwith primers 2HMR8 (5′-CGCGGATCCGTGCAGTCCTGCTCCGCCTACGGC-3′) (SEQ ID NO:214) and 2HMR11 (5′-CCGAATTCTTATGCTGGTACTTCAACTATTTCAAAGTAG-3′) (SEQ IDNO: 215), digested with BamHI and EcoRI, and cloned in frame downstreamof the Glutathion S-Transferase ORF, between the BamHI and EcoRI sitesof the pGEX-2T prokaryotic expression vector (Amersham PharmaciaBiotech, Saclay, France). GST-THAP1 fusion protein encoded by plasmidpGEX-2T-THAP1 and control GST protein encoded by plasmid pGEX-2T, werethen expressed in E. coli DH5α and purified by affinity chromatographywith glutathione sepharose according to supplier's instructions(Amersham Pharmacia Biotech). The yield of proteins used in GSTpull-down assays was determined by SDS-Polyarylamide Gel Electrophoresis(PAGE) and Coomassie blue staining analysis.

In vitro-translated SLC/CCL21 was generated with the TNT-coupledreticulocyte lysate system (Promega, Madison, Wis., USA) using astemplate pGBKT7-SLC/CCL21 vector (see Example 15). 25 μl of ³⁵S-labelledwild-type SLC/CCL21 was incubated with immobilized GST-THAP1 or GSTproteins overnight at 4° C., in the following binding buffer: 10 mMNaPO4 pH 8.0, 140 mM NaCl, 3 mM MgCl2, 1 mM dithiothreitol (DTT), 0.05%NP40, and 0.2 mM phenylmethyl sulphonyl fluoride (PMSF), 1 mM NaVanadate, 50 mM β Glycerophosphate, 25 μg/ml chimotrypsine, 5 μg/mlaprotinin, 10 μg/ml Leupeptin. Beads were then washed 5 times in 1 mlbinding buffer. Bound proteins were eluted with 2× Laenmmli SDS-PAGEsample buffer, fractionated by 10% SDS-PAGE and visualized byfluorography using Amplify (Amersham Pharmacia Biotech). As expected,GST/THAP1 interacted with SLC/CCL21 (FIG. 13). In contrast, SLC/CCL21failed to interact with GST beads.

Example 17 Identification of the THAP1-Binding Domain of Human ChemokineSLC/CCL21

To determine the THAP1-binding site on human chemokine SLC/CCL21, aSLC/CCL21 deletion mutant (SLC/CCL21ΔCOOH) lacking the SLC-specificbasic carboxy-terminal extension (amino acids 102-134; GenBank AccessionNumber NP_(—)002980) was generated. This SLC/CCL21ΔCOOH mutant, whichretains the CCR7 chemokine receptor binding domain of SLC/CCL21 (aminoacids 24-101), was used both in yeast two-hybrid assays with THAP1 baitand in in vitro GST-pull down assays with GST-THAP1.

For two-hybrid assays, yeast cells were cotransformed with BD7-THAP1 andAD7-SLC/CCL21 or AD7-SLC/CCL21ΔCOOH expression vectors. AD7-SLC/CCL21 orAD7-SLC/CCL21ΔCOOH expression vectors were generated by subcloning BamHIfragment (encoding SLC amino acids 24-134) or BamHI-PstI fragment(encoding SLC amino acids 24-102) from pGKT7-SLC/CCL21 (see example 15)into pGADT7 expression vector (Clontech). Transformants were selected onmedia lacking histidine and adenine. FIG. 13 shows that both theSLC/CCL21 wild type and the SLC/CCL21ΔCOOH deletion mutants could bindto THAP1. Identical results were obtained by cotransformation ofAD7-THAP1 with BD7-SLC/CCL21 or BD7-SLC/CCL21ΔCOOH.

GST pull down assays, using in vitro-translated SLC/CCL21ΔCOOH,generated with the TNT-coupled reticulocyte lysate system (Promega,Madison, Wis., USA) using as template pGBKT7-SLC/CCL21ΔCOOH, wereperformed as described in Example 16. FIG. 13 shows that both theSLC/CCL21 wild type and the SLC/CCL21ΔCOOH deletion mutants could bindto THAP1.

Example 18A Preparation of THAP1/Fc Fusion Proteins

This example describes preparation of a fusion protein comprising THAP1or the SLC/CCL21 chemokine-binding domain of THAP1 fused to an Fc regionpolypeptide derived from an antibody. An expression vector encoding theTHAP1/Fc fusion protein is constructed as follows.

Briefly, the full length coding region of human THAP1 (SEQ ID NO: 3;amino acids −1 to 213) or the SLC/CCL21 chemokine-binding domain ofhuman THAP1 (SEQ ID NO: 3; amino acids −143 to 213) is amplified by PCR.The oligonucleotides employed as 5′ primers in the PCR contain anadditional sequence that adds a Not I restriction site upstream. The 3′primer includes an additional sequence that encodes the first two aminoacids of an Fc polypeptide, and a sequence that adds a BglII restrictionsite downstream of the THAP1 and Fc sequences.

A recombinant vector containing the human THAP1 cDNA is employed as thetemplate in the PCR, which is conducted according to conventionalprocedures. The amplified DNA is then digested with Not I and Bgl II,and the desired fragments are purified by electrophoresis on an agarosegel.

A DNA fragment encoding the Fc region of a human IgG1 antibody isisolated by digesting a vector containing cloned Fc-encoding DNA withBgl II and Not I. Bgl II cleaves at a unique Bgl II site introduced nearthe 5′ end of the Fc-encoding sequence, such that the Bgl II siteencompasses the codons for amino acids three and four of the Fcpolypeptide. Not I cleaves downstream of the Fc-encoding sequence. Thenucleotide sequence of cDNA encoding the Fc polypeptide, along with theencoded amino acid sequence, can be found in International PublicationNo: WO93/10151, incorporated herein by reference in its entirety.

In a three-way ligation, the above-described THAP1 (or SLC/CCL21chemokine-binding domain of THAP1) -encoding DNA and Fc-encoding DNA areinserted into an expression vector that has been digested with Not I andtreated with a phosphatase to minimize recircularization of any vectorDNA without an insert. An example of a vector which can be used ispDC406 (described in McMahan et al., EMBO J. 10:2821, 1991), which is amammalian expression vector that is also capable of replication in E.coli.

E. coli cells are then transfected with the ligation mixture, and thedesired recombinant vectors are isolated. The vectors encode aminoacids-1 to 213 of the THAP1 sequence (SEQ ID NO: 3) or amino acids-143to 213 of the THAP1 sequence of (SEQ ID NO: 3), fused to the N-terminusof the Fc polypeptide. The encoded Fc polypeptide extends from theN-terminal hinge region to the native C-terminus, i.e., is anessentially full-length antibody Fc region.

CV-1/EBNA-1 cells are then transfected with the desired recombinantisolated from E. coli. CV-11/EBNA-1 cells (ATCC CRL 10478) can betransfected with the recombinant vectors by conventional procedures. TheCVI-EBNA-1 cell line was derived from the African Green Monkey kidneycell line CV-1 (ATCC CCL 70), as described by McMahan et al. (1991).EMBO J. 10:2821. The transfected cells are cultured to allow transientexpression of the THAP1/Fc or SLC/CCL21 chemokine-binding domain ofTHAP1/Fc fusion proteins, which are secreted into the culture medium.The secreted proteins contain the mature form of THAP1 or the SLC/CCL21chemokine-binding domain of THAP1, fused to the Fc polypeptide. TheTHAP1/Fc and SLC/CCL21 chemokine-binding domain of THAP1/Fc fusionproteins are believed to form dimers, wherein two such fusion proteinsare joined by disulfide bonds that form between the Fc moieties thereof.The THAP1/Fc and SLC/CCL21 chemokine-binding domain of THAP1/Fc fusionproteins can be recovered from the culture medium by affinitychromatography on a Protein A-bearing chromatography column.

Example 18B Preparation of THAP1/IgG1-Fc Fusion Proteins

This example describes preparation of a fusion protein comprising THAP1or the SLC/CCL21 chemokine-binding domain of THAP1 fused to an Fc regionpolypeptide derived from an antibody. An expression vector encoding theTHAP1/IgG1-Fc fusion protein was derived from a pCDM8 expression vectorencoding L-selectin-IgG1 fusion proteins (recombinant chimeric moleculescontaining extracellular regions of L-selectin coupled to the hinge,CH2, and CH3 regions of human IgG1) as described in Aruffo, A., et al.,Cell, 67:35, 1991, and Walz, G., et al., Science, 250:1132, 1990, thedisclosures of which are incorporated herein by reference in theirentireties. The nucleotide sequence of cDNA encoding the IgG1-Fcpolypeptide, along with the encoded amino acid sequence is described inInternational Publication No. WO93/10151, the disclosure of which isincorporated herein by reference in its entirety.

Briefly, the full length coding region of human THAP1 (SEQ ID NO: 3;amino acids −2 to 213) or the SLC/CCL21 chemokine-binding domain ofhuman THAP1 (CBD/THAP1, SEQ ID NO: 3; amino acids −140 to 213) wereamplified by PCR with primers THAP1-XhoI-5′(5′-CCGCTCGAGGTGCAGTCCTGCT-3′) (SEQ ID NO: 264) and THAP1-BamHI-3′(5′-CGGGATCCGCTGGTACTTCAACTATTTCA-3′) (SEQ ID NO: 265), or primersCBD/THAP1-XhoI-5′ (5′-CCGCTCGAGGATACAATGCACC-3′) (SEQ ID NO: 266) andCBD/THAP1-BamHI-3′ (5′-GCGGGATCCGCTGGTACTTCAACTATTTCAAAG-3′) (SEQ ID NO:267), respectively. A recombinant vector containing the human THAP1 cDNA(see example 7) was employed as the template in the PCR, which wasconducted according to conventional procedures. The amplified DNAs werethen digested with Xho I and BamH I and the desired fragments werepurified by electrophoresis on an agarose gel. The resulting Xho I-BamHI fragments were then used to replace the Xho I-BamH I fragment encodingL-selectin in the plasmid pCDM8-L-selectin-IgG1 (Aruffo, A., et al.,Cell, 67:35, 1991; Walz, G., et al., Science, 250:1132, 1990). Therecombinant vectors thus obtained, pCDM8-THAP1-IgG1 andpCDM8-CBD/THAP1-IgG1, encode amino acids-2 to 213 of the THAP1 sequence(SEQ ID NO: 3) or amino acids-140 to 213 of the THAP1 sequence of (SEQID NO: 3), fused to the N-terminus of the IgG1-Fc polypeptide. Becausethe encoded IgG1-Fc region of the fusion polypeptides extend from theN-terminal hinge region to the native C-terminus, the IgG1-Fc region isessentially a full-length antibody Fc region.

In addition to fusion the IgG1-Fc region to THAP1 and CBD/THAP1, thesignal peptide of immunoglobulin kappa light chain was fused to theN-terminus of each of these proteins. A nucleic acid encoding the signalpeptide was obtained by using PCR to amplify a SalI-XhoI signal peptidecassette in two steps. In the first step, the oligonucleotide psignal5′(5′-CCGCTCGAG CCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACCTCGAGATT-3′) (SEQ ID NO: 268), which encodes the 21amino acids of the immunoglobulin kappa chain signal peptide fromplasmid vector pSecTag2 (Invitrogen) was synthesized and then used as atemplate for PCR with primers psignal-SalI 5′(5′-TAGGGTCGACGCCACCATGGAGACAG-3′) (SEQ ID NO: 269) and psignalXhol 3′(5′-CCGCTCGAGGTCACCAGTGGA-3′) (SEQ ID NO: 270). The product of the PCRreaction was digested with Sal I and Xho I and ligated into the Xho Isite of plasmids pCDM8-THAP1-IgG1 and pCDM8-CBD/THAP1-IgG1 to obtainexpression vectors pCDM8-SS-THAP1-IgG1 and pCDM8-SS-CBD/THAP1-IgG1.These plasmids were then transfected in COS cells or CV-1/EBNA-1 cells(ATCC CRL 10478), as previously described (Seed, B., et al., Proc. Natl.Acad. Sci., U.S.A., 84:3365, 1987; Aruffo, A., Current Protocols InMolecular Biology, eds. Ausubel, F. M., et al, 16:13.1, GreenePublishing Associates and Wiley-Interscience, New York, N.Y., 1992, thedisclosures of which are incorporated herein by reference in theirentireties). The CVI-EBNA-1 cell line was derived from the African GreenMonkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan etal. (1991). EMBO J. 10:2821, the disclosure of which is incorporated byreference herein in its entirety. The transfected cells were cultured toallow transient expression of the THAP1/Fc or SLC/CCL21chemokine-binding domain of THAP1/Fc fusion proteins, which weresecreted into the culture medium. The proteins that were secretedcontain the mature form of THAP1 or the SLC/CCL21 chemokine-bindingdomain of THAP1, fused to the Fc polypeptide. Although not bound bytherory, the THAP1/Fc and SLC/CCL21 chemokine-binding domain of THAP1/Fcfusion proteins are believed to form dimers, wherein two such fusionproteins are joined by disulfide bonds that form between the Fc moietiesthereof. The THAP1/Fc and SLC/CCL21 chemokine-binding domain of THAP1/Fcfusion proteins were recovered from the culture medium by affinitychromatography on a Protein A-bearing chromatography column.

Example 19 The THAP Domain Defines a Family of Nuclear Factors

To determine the subcellular localization of the different human THAPproteins, a series of GFP-THAP expression constructs were transfectedinto primary human endothelial cells. In agreement with the possiblefunctions of THAP proteins as DNA-binding factors, we found that all thehuman THAP proteins analyzed (THAP0, 1, 2, 3, 6, 7, 8, 10, 11) localizepreferentially to the cell nucleus (FIG. 14). In addition to theirdiff-use nuclear localization, some of the THAP proteins also exhibitedassociation with distinct subnuclear structures: the nucleolus for THAP2and THAP3, and punctuate nuclear bodies for THAP7, THAP8 and THAP11.Indirect immunofluorescence microscopy with anti-PML antibodies revealedthat the THAP8 and THAP11 nuclear bodies colocalize with PML-NBs.Although the THAP7 nuclear bodies often appeared in close associationwith the PML-NBs, they never colocalized.

Analysis of the subcellular localization of the GFP-THAP fusion proteinswas performed as described above (Example 3). The GFP-THAP constructswere generated as follows: the human THAP0 coding region was amplifiedby PCR from Hevec cDNA with primers THAP0-1(5′-GCCGAATTCATGCCGAACTTCTGCGCTGCCCCC-3′) (SEQ ID NO: 216) and THAP0-2(5′-CGCGGATCCTTAGGTTATTTTCCACAGTTTCGGAATTATC-3′) (SEQ ID NO: 217),digested with EcoRI and BamHI, and cloned in the same sites of thepEGFP-C2 vector, to generate pEGFPC2-THAP0; the coding region of humanTHAP2, 3, 7, 6 and 8 were amplified by PCR respectively from Image cloneNo: 3606376 with primers THAP2-1(5′-GCGCTGCAGCAAGCTAAATTTAAATGAAGGTACTCTTGG-3′) (SEQ ID NO: 218) andTHAP2-2 (5′-GCGAGATCTGGGAAATGCCGACCAATTGCGCTGCG-3′) (SEQ ID NO: 219)digested with BglII and PstI, from Image clone No: 4813302 and No:3633743 with primers THAP3-1 (5′-AGAGGATCCTTAGCTCTGCTGCTCTGGCCCAAGTC-3′)(SEQ ID NO: 220) THAP3-2 (5′-AGAGAATTCATGCCGAAGTCGTGCGCGGCCCG-3′) (SEQID NO: 221) and primers THAP7-1 (5′-GCGGAATTCATGCCGCGTCACTGCTCCGCCGC-3′)(SEQ ID NO: 222) THAP7-2 (5′-GCGGGATCCTCAGGCCATGCTGCTGCTCAGCTGC-3′) (SEQID NO: 223), digested with EcoRI and BamHI, from Image clone No: 757753with primers THAP6-1 (5′-GCGAGATCTCGATGGTGAAATGCTGCTCCGCCATTGGA-3′) (SEQID NO: 224) and THAP6-2 (5′-GCGGGATCCTCATGAAATATAGTCCTGTTCTATGCTCTC-3′)(SEQ ID NO: 225) digested with BglII and BamHI, and from Image clone No:4819178 with primers THAP8-1 (5′-GCGAGATCTCGATGCCCAAGTACTGCAGGGCGCCG-3′)(SEQ ID NO: 226) and THAP8-2(5′-GCGGAATTCTTATGCACTGGGGATCCGAGTGTCCAGG-3′) (SEQ ID NO: 227), digestedwith BglII and EcoRI and cloned in frame downstream of the EnhancedGreen Fluorescent Protein (EGFP) ORF in pEGFPC2 vector (Clontech)digested with the same enzymes to generate pEGFPC2-THAP2, -THAP3,-THAP7, -THAP6 and -THAP8; the human THAP10 and THAP11 coding regionwere amplified by PCR from Hela cDNA respectively with primers THP10-1(5′-GCGGAATTCATGCCGGCCCGTTGTGTGGCCGC-3′) (SEQ ID NO: 228) THAP10-2(5′-GCGGGATCCTTAACATGTTTCTTCTTTCACCTGTACAGC-3′) (SEQ ID NO: 229)digested with EcoRI and BamHI, and with primers THAP11-1(5′-GCGAGATCTCGATGCCTGGCTTTACGTGCTGCGTGC-3′) (SEQ ID NO: 230) andTHAP11-2 (5′-GCGGAATTCTCACATTCCGTGCTTCTTGCGGATGAC-3′) (SEQ ID NO: 231),digested with BglII and EcoRI, cloned in the same sites of the pEGFP-C2vector, to generate pEGFPC2-THAP10 and -THAP11.

Example 20 The THAP Domain Shares Structural Similarities with theDNA-Binding Domain of Nuclear Hormone Receptors

In an effort to model the three-dimensional structure of the THAPdomain, we searched the PDB crystallographic database. As sequencehomology detection is more sensitive and selective when aided bysecondary structure information, structural homologs of the THAP domainof human THAP1 were searched using the SeqFold threading program(Olszewski et al. (1999) Theor. Chem. Acc. 101, 57-61) which combinessequence and secondary structure alignment. The crystallographicstructure of the thyroid hormone receptor β DBD (PDB code: 2NLL) gavethe best score of the search and we used the resulting structuralalignment, displayed in FIG. 15A, to derive a homology-based model ofthe THAP domain from human THAP1 (FIG. 15B). Note that the distributionof Cys residues in the THAP domain does not fully match that of thethyroid hormone receptor β DBD (FIG. 15A) and hence cannot allow theformation of the two characteristic ‘C4-type’ Zn-fingers (redcolor-coding in FIG. 15A). However, a network of stacking interactionsbetween aromatic/hydrophobic residues or aliphatic parts of lysineside-chains ensures the stability of the structure of the THAP domain(cyan color-coding in FIGS. 15A and 15B). Interestingly the samethreading method applied independently to the Drosophila P-elementtransposase DBD identified the crystallographic structure of theglucocorticoid receptor DBD (PDB code: IGLU) as giving the best score.In the same way, we used the resulting structural alignment, displayedin FIG. 15D, to build a model of the transposase DBD (FIG. 15C). Notethe presence of an hydrophobic core equivalent to that of the THAPdomain (cyan color-coding in FIGS. 15C and 15D). All the DNA-bindingdomains of the nuclear receptors fold into a typical pattern which ismainly based on two interacting α-helices, the first one inserting intothe target DNA major groove. Our threading and modeling results indicatethat the THAP domain and the D. melanogaster P-element transposase DBDlikely share a common topology which is similar to that of the DBD ofnuclear receptors.

Molecular modeling was performed using the InsightII, SeqFold, Homologyand Discover modules from the Accelrys (San Diego, Calif.) molecularmodeling software (version 98), run on a Silicon Graphics O2workstation. Optimal secondary structure prediction of the query proteindomains was ensured by the DSC method within SeqFold. Thethreading-derived secondary structure alignments was used as input forhomology-modeling, which was performed according to a previouslydescribed protocol (Manival et al. (2001) Nucleic Acids Res 29:2223-2233). The validity of the models was checked both by Ramachandrananalysis and folding consistency verification as previously reported(Manival et al. (2001) Nucleic Acids Res 29 :2223-2233).

Example 21 Homodimerization Domain of Human THAP1

To identify the sequences mediating homodimerization of THAP1, a seriesof THAP1 deletion constructs was generated as described in Example 7.

Two-hybrid interaction between THAP1 mutants and THAP1 wild type wastested by cotransformation of AH109 with pGADT7-THAP1-C1, —C2, —C3, —N1,—N2 or —N3 and pGBKT7-THAP1 wild-type and selection of transformants byHis and Ade double auxotrophy according to manufacturer's instructions(MATCHMAKER two-hybrid system 3, Clontech). Positive two-hybridinteraction with THAP1 wild type was observed with mutants THAP1—C1,—C2, —C3, -and —N3 but not with mutants THAP1-N1 and —N2, suggesting theTHAP1 homodimerization domain is found between THAP1 residues 143 and192 (FIG. 16A).

To confirm the results obtained in yeast, THAP1 mutants were also testedin in vitro GST pull down assays. Wild type THAP1 expressed as aGST-tagged fusion protein and immobilized on glutathione sepharose (asdescribed in example 16), was incubated with radiolabeled in vitrotranslated THAP1 mutants. In vitro-translated THAP1 mutants weregenerated with the TNT-coupled reticulocyte lysate system (Promega,Madison, Wis., USA) using pGADT7-THAP1-C1, —C2, —C3, —N1, —N2 or —N3vector as template. 25 μl of each ³⁵S-labelled THAP1 mutant wasincubated with immobilized GST or GST-THAP1 wild-type protein overnightat 4° C., in the following binding buffer: 10 mM NaPO4 pH 8.0, 140 mMNaCl, 3 mM MgCl2, 1 mM dithiothreitol (DTT), 0.05% NP40, and 0.2 mMphenylmethyl sulphonyl fluoride (PMSF), 1 mM Na Vanadate, 50 mM βGlycerophosphate, 25 μg/ml chimotrypsine, 5 μg/ml aprotinin, 10 μg/mlLeupeptin. Beads were then washed 5 times in 1 ml binding buffer. Boundproteins were eluted with 2× Laemmli SDS-PAGE sample buffer,fractionated by 10% SDS-PAGE and visualized by fluorography usingAmplify (Amersham Pharmacia Biotech). As expected, THAP1—C1, —C2, —C3,-and —N3 interacted with GST/THAP1 (FIG. 16B). In contrast, THAP1-N1 and—N2 failed to interact with GST/THAP1 beads. Therefore, essentiallyidentical results were obtained with the two THAP1/THAP1 interactionsassays: the THAP1 homodimerization domain of THAP1 is found betweenresidues 143 and 192 of human THAP1.

Example 22 Alternatively Spliced Isoform of Human THAP1

The two distinct THAP1 cDNAs, THAP1a and THAP1b have been discovered(FIG. 17A). These splice variants, were amplified by PCR from HEVEC cDNAwith primers 2HMR 10(5′-CCGAATTCAGGATGGTGCAGTCCTGCTCCGCCT-3′) (SEQ IDNO: 232) and 2HMR9 (5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQID NO: 233), digested with EcoRI and BamHI, and cloned in frame upstreamof the Enhanced Green Fluorescent Protein (EGFP) ORF in pEGFP.N3 vector(Clontech) to generate pEGFP.N3-THAP1a and pEGFP-THAP1b. DNA sequencingrevealed that THAP1b cDNA isoform lacks exon 2 (nucleotides 273-468) ofthe human THAP1 gene (FIG. 17B). This alternatively spliced isoform ofhuman THAP1 (˜2 kb mRNA) was also observed in many other tissues byNorthern blot analysis (see FIG. 2). The THAP1a/GFP and THAP1b/GFPexpression constructs were then transfected into COS 7 cells (ATCC) andexpression of the fusion proteins was analyzed by western blotting withanti-GFP antibodies. The results are shown in FIG. 17C whichdemonstrates that the second isoform of human THAP1 (THAP1b) encodes atruncated THAP1 protein (THAP1 C3) lacking a substantial portion of theamino terminus (amino acids 1-142 of SEQ ID NO: 3).

Example 23 High Throughput Screening Assay for Modulators of THAP FamilyPolypeptide Pro-Apoptotic Activity

A high throughput screening assay for molecules that abrogate orstimulate THAP-family polypeptide proapoptotic activity was developed,based on serum-withdrawal induced apoptosis in a 3T3 cell line withtetracycline-regulated expression of a THAP family polypeptide.

In a preferred example, the THAP1 cDNA with an in-frame myc tagsequence, was amplified by PCR using pGBKT7-THAP1 as a template withprimers myc.BD7 (5′-GCGCTCTAGAGCCATCATGGAGGAGCAGAAGCTGATC-3′) (SEQ IDNO: 234) and 2HMR15 (5′-GCGCTCTAGATTATGCTGGTACTTCAACTATTTCAAAGTAG-3′)(SEQ ID NO: 235), and cloned downstream of a tetracycline regulatedpromoter in plasmid vector pTRE (Clontech, Palo Alto, Calif.), using XbaI restriction site, to generate plasmid pTRE-mycTHAP1. To establish3T3-TO-mycTHAP1 stable cell lines, mouse 3T3-TO fibroblasts (Clontech)were seeded at 40 to 50% confluency and co-transfected with the pREP4plasmid (Invitrogen), which contains a hygromycin B resistance gene, andthe mycTHAP1 expression vector (pTRE-mycTHAP1) at 1:10 ratio, usingLipofectamine Plus reagent (Life Technologies) according to supplier'sinstructions. Transfected cells were selected in medium containinghygromycin B (250 U/ml; Calbiochem) and tetracycline (2 ug/ml; Sigma).Several resistant colonies were picked and analyzed for the expressionof mycTHAP1 by indirect immunofluorescence using anti-myc epitopemonoclonal antibody (mouse IgG1, 1/200, Clontech). A stable 3T3-TO cellline expressing mycTHAP1 (3T3-TO-mycTHAP1) was selected and grown inDulbecco's Modified Eagle's Medium supplemented with 10% Fetal CalfSerum, 1% Penicillin-streptomycin (all from Life Technologies, GrandIsland, N.Y., USA) and tetracycline (2 ug/ml; Sigma). Induction of THAP1expression into this 3T3-TO-mycTHAP1 cell line was obtained 48 h afterremoval of tetracycline in the complete medium.

A drug screening assay using the 3T3-TO-mycTHAP1 cell line can becarried out as follows. 3T3-TO-mycTHAP1 cells are plated in 96- or384-wells microplates and THAP1 expression is induced by removal oftetracycline in the complete medium. 48 h later, the apoptotic responseto serum withdrawal is assayed in the presence of a test compound,allowing the identification of test compounds that either enhance orinhibit the ability of THAP1 polypeptide to induce apoptosis. Serumstarvation of 3T3-TO-mycTHAP1 cells is induced by changing the medium to0% serum, and the amount of cells with apoptotic nuclei is assessed 24 hafter induction of serum starvation by TUNEL labeling in 96- or384-wells microplates. Cells are fixed in PBS containing 3.7%formaldehyde and permeabilized with 0.1% Triton-X100, and apoptosis isscored by in situ TUNEL (terminal deoxynucleotidyl transferase-mediateddUTP nick end labeling) staining of apoptotic nuclei for 1 hr at 37° C.using the in situ cell death detection kit, TMR red (Roche Diagnostics,Meylan, France). The intensity of TMR red fluorescence in each well isthen quantified to identify test compounds that modify the fluorescencesignal and thus either enhance or inhibit THAP1 pro-apoptotic activity.

Example 24 High Throughput Two-Hybrid Screening Assay for Drugs thatModulate THAP-Family Polypeptide/THAP-Family Target Protein Interaction

To identify drugs that modulate THAP1/Par4 or THAP1/SLC interactions, atwo-hybrid based high throughput screening assay can be used.

As described in Example 17, AH109 yeast cells (Clontech) cotransformedwith plasmids pGBKT7-THAP1 and pGADT7-Par4 or pGADT7-SLC can be grown in384-well plates in selective media lacking histidine and adenine,according to manufacturer's instructions (MATCHMAKER two-hybrid system3, Clontech).

Growth of the transformants on media lacking histidine and adenine isabsolutely dependent on the THAP1/Par4 or THAP1/SLC two-hybridinteraction and drugs that disrupt THAP1/Par4 or THAP1/SLC binding willtherefore inhibit yeast cell growth.

Small molecules (5 mg ml⁻¹ in DMSO; Chembridge) are added by usingplastic 384-pin arrays (Genetix). The plates are incubated for 4 to 5days at 30° C., and small molecules which inhibit the growth of yeastcells by disrupting THAP1/Par4 or THAP1/SLC two-hybrid interaction areselected for further analysis.

Example 25 High Throughput in vitro Assay to Identify Inhibitors ofTHAP-Family Polypeptide/THAP-Family Protein Target Interaction

To identify small molecule modulators of TRAP function, ahigh-throughput screen based on fluorescence polarization (FP) is usedto monitor the displacement of a fluorescently labelled THAP1 proteinfrom a recombinant glutathione-S-transferase (GST)-THAP binding domainof Par4 (Par4DD) fusion protein or a recombinant GST-SLC/CCL21 fusionprotein.

Assays are carried out essentially as in Degterev et al, Nature CellBiol. 3: 173-182 (2001) and Dandliker et al, Methods Enzymol. 74: 3-28(1981). The assay can be calibrated by titrating a THAP1 peptidelabelled with Oregon Green with increasing amounts of GST-Par4DD orGST-SLC/CCL21 proteins. Binding of the peptide is accompanied by anincrease in polarization (mP, millipolarization).

THAP1 and PAR4 polypeptides and GST-fusions can be produced aspreviously described. The THAP1 peptide was expressed and purified usinga QIAexpressionist kit (Qiagen) according to the manufacturer'sinstructions. Briefly, the entire THAP1 coding sequence was amplified byPCR using pGBKT7-THAP1 as a template with primers 2HMR8(5′-CGCGGATCCGTGCAGTCCTGCTCCGCCTACGGC-3′) (SEQ ID NO: 236) and 2HMR9(5′-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-3′) (SEQ ID NO: 237), andcloned into the BamHI site of pQE30 vector (Qiagen). The resultingpQE30-HisTHAP1 plasmid was transformed in E.coli strain M15 (Qiagen). 6×His-tagged-THAP1 protein was purified from inclusion bodies on aNi-Agarose column (Qiagen) under denaturing conditions, and the eluatewas used for in vitro interaction assays. To produce GST-Par4DD fusionprotein, Par4DD (amino acids 250-342) was amplified by PCR with primersPar4. 10 (5′-GCCGGATCCGGGTTCCCTAGATATAACAGGGATGCAA-3′) (SEQ ID NO: 238)and Par4.5 (5′-GCGGGATCCCTCTACCTGGTCAGCTGACCCACAAC-3′) (SEQ ID NO: 239),and cloned in frame downstream of the Glutathione S-Transferase (GST)ORF, into the BamHI site of the pGEX-2T prokaryotic expression vector(Amersham Pharmacia Biotech, Saclay, France). Similarly, to produceGST-SLC/CCL21 fusion protein, the mature form of human SLC/CCL21 (aminoacids 24-134) was amplified by PCR with primers hSLCbam.5′(5′-GCGGGATCCAGTGATGGAGGGGCTCAGGACTGTTG-3′) (SEQ ID NO: 240) andhSLCbam.3′ (5′-GCGGGATCCCTATGGCCCTTTAGGGGTCTGTGACC-3′) (SEQ ID NO: 241),digested with BamHI and inserted into the BamHI cloning site of thepGEX-2T vector. GST-Par4DD (amino acids 250-342) and GST-SLC/CCL21(amino acids 24-134) fusion proteins were expressed in E. Coli DH5α(supE44, DELTAlacU169 (801acZdeltaM15), hsdR17, recA1, endA1, gyrA96,thi1, relA 1) and purified by affinity chromatography with glutathionesepharose according to supplier's instructions (Amersham PharmaciaBiotech).

For screening small molecules, THAP1 peptide is labelled withsuccinimidyl Oregon Green (Molecular Probes, Oregon) and purified byHPLC. 33 nM labeled THAP1 peptide, 2 μM GST-Par4DD or GST-SLC/CCL21protein, 0.1% bovine gamma-globulin (Sigma) and 1 mM dithiothreitolmixed with PBS, pH 7.2 (Gibco), are added to 384-well black plates (LabSystems) with Multidrop (Lab Systems). Small molecules (5 mg ml⁻¹ inDMSO; Chembridge) are transferred by using plastic 384-pin arrays(Genetix). The plates are incubated for 1-2 hours at 25° C., and FPvalues are determined with an Analyst plate reader (LJL Biosystems).

Example 26 High Throughput Chip Assay to Identify Inhibitors ofTHAP-Family Polypeptide/THAP-Family Protein Target Interaction

A chip based binding assay Degterev et al, (2001) Nature Cell Biol. 3:173-182 using unlabelled THAP and THAP-family target protein may be usedto identify molecules capable of interfering with THAP-family andTHAP-family target interactions, providing high sensitivity and avoidingpotential interference from label moieties. In this example, the THAP1binding domain of Par4 protein (Par4DD) or SLC/CCL21 is covalentlyattached to a surface-enhanced laser desorption/ionization (SELDI) chip,and binding of unlabelled THAP1 protein to immobilized protein in thepresence of a test compound is monitored by mass spectrometry.

Recombinant THAP1 protein, GST-Par4DD and GST-SLC/CCL21 fusion proteinsare prepared as described in Example 25. Purified recombinant GST-Par4DDor GST-SLC/CCL21 protein is coupled through its primary amine to SELDIchip surfaces derivatized with cabonyldiimidazole (Ciphergen). THAP1protein is incubated in a total volume of 1 μl for 12 hours at 4° C. ina humidified chamber to allow binding to each spot of the SELDI chip,then washed with alternating high-pH and low-pH buffers (0.1M sodiumacetate containing 0.5M NaCl, followed by 0.01 M HEPES, pH 7.3). Thesamples are embedded in an alpha-cyano-4-hydroxycinnamic acid matrix andanalysed for mass by matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) mass spectrometry. Averages of 100 lasershots at a constant setting are collected over 20 spots in each sample.

Example 27 High Throughput Cell Assay to Identify Inhibitors ofTHAP-Family Polypeptide/THAP-Family Protein Target Interaction

A fluorescence resonance energy transfer (FRET) assay is carried outbetween THAP-1 and PAR4 or SLC/CCL21 proteins fused with fluorescentproteins. Assays can be carried out as in Majhan et al, NatureBiotechnology 16: 547-552 (1998) and Degterev et al, Nature Cell Biol.3: 173-182 (2001).

THAP-1 protein is fused to cyan fluorescent protein (CFP) and PAR4 orSLC/CCL21 protein is fused to yellow fluorescent protein (YFP). Vectorscontaining THAP-family and THAP-family target proteins can beconstructed essentially as in Majhan et al (1998). A THAP-1-CFPexpression vector is generated by subcloning a THAP-1 cDNA into thepECFP-N1 vector (Clontech). PAR4-YFP or SLC/CCL21-CYP expression vectorsare generated by subcloning a PAR4 or a SLC/CCL21 cDNA into the pEYFP-N1vector (Clontech).

Vectors are cotransfected to HEK-293 cells and cells are treated withtest compounds. HEK-293 cells are transfected with THAP-1-CFP andPAR4-YFP or SLC/CCL21-YFP expression vectors using Lipofect AMINE Plus(Gibco) or TransLT-1 (PanVera). 24 hours later cells are treated withtest compounds and incubated for various time periods, preferably up to48 hours. Cells are harvested in PBS, optionally supplemented with testcompound, and fluorescence is determined with a C-60 fluorimeter (PTI)or a Wallac plate reader. Fluorescence in the samples separatelyexpressing THAP-1-CFP and PAR4-YFP or SLC/CCL21-YFP is added togetherand used to estimate the FRET value in the absence of THAP-1/PAR4 orTHAP1/SLC/CCL21 binding.

The extent of FRET between CFP and YFP is determined as the ratiobetween the fluorescence at 527 nm and that at 475 nm after excitationat 433 nm. The cotransfection of THAP-1 protein and PAR4 or SLC/CCL21protein results in an increase of FRET ratio over a reference FRET ratioof 1.0 (determined using samples expressing the proteins separately). Achange in the FRET ratio upon treatmemt with a test compound (over thatobserved after cotransfection in the absence of a test compound)indicates a compound capable of modulating the interaction of the THAP-1protein and the PAR4 or the SLC/CCL21 protein.

Example 28 In vitro Assay to Identify THAP-Family Polypeptide DNATargets

DNA binding specificity of THAP1 was determined using a randomoligonucleotide selection method allowing unbiased analysis of bindingsites selected by the THAP domain of the THAP1 protein from a randompool of possible sites. The method was carried out essentially asdescribed in Bouvet (2001) Methods Mol Biol. 148:603-10. Also, seePollack and Treisman (1990) Nuc. Acid Res. 18:6197-6204; Blackwell andWeintraub, (1990) Science 250: 1104-1110; Ko and Engel, (1993) Mol.Cell. Biol. 13:4011-4022; Merika and Orkin, (1993) Mol. Cell. Biol. 13:3999-4010; and Krueger and Morimoto, (1994) Mol. Cell. Biol.14:7592-7603), the disclosures of which are incorporated herein byreference in their entireties.

Recombinant THAP Domain Expression and Purification

A cDNA fragment encoding the THAP domain of human THAP-1 (amino acids1-90, SEQ ID NO: 3) was cloned by PCR using as a template pGADT7-THAP-1(see Example 4) with the following primers5′-GCGCATATGGTGCAGTCCTGCTCCGCCTACGGC-3′ (SEQ ID NO: 242) and5′-GCGCTCGAGTTTCTTGTCATGTGGCTCAGTACAAAG-3′ (SEQ ID NO: 243). The PCRproduct was cloned as a NdeI-XhoI fragment into pET-21c prokaryoticexpression vector (Novagen) in frame with a sequence encoding a carboxyterminal His tag, to generate pET-21c-THAP.

For the expression of THAP-His6, pET-21c-THAP was transformed intoEscherichia coli strain BL-21 pLysS. Bacteria were grown at 37° C. to anoptical density at 600 nm of 0.6 and expression of the protein wasinduced by adding isopropyl-β-D-thiogalactoside (Sigma) at a finalconcentration of 1 mM and incubation was continued for 4 hours.

The cells were collected by centrifugation and resuspended in ice coldof buffer A (50 mM sodium-phosphate pH 7.5, 300 mM NaCl, 0.1%β-mercaptoethanol, 10 mM Imidazole). Cells were lysed by sonication andthe lysate was cleared by centrifugation at 12000 g for 45 min. Thesupernatant was loaded onto a Ni-NTA agarose column (Quiagen)equilibrated in buffer A. After washing with buffer A and Buffer A with40 mM Imidazole, the protein was eluted with buffer B (same as A with0.05% β-mercaptoethanol and 250 mM Imidazole).

Fractions containing THAP-His6 were pooled and applied to a Superdex 75gel filtration column equilibrated in Buffer C (Tris-HCl 50 mM pH 7.5,150 mM NaCl, 1 mM DTT). Fractions containing the THAP-His6 protein werepooled, concentrated with YM-3 Amicon filter devices and stored at 4° C.or frozen at −80° C. in buffer C containing 20% glycerol. The purity ofthe sample was assessed by SDS-Polyarylamide Gel Electrophoresis (PAGE)and Coomassie blue staining analysis. The structural integrity of theprotein preparation was checked by ESI mass spectrometry and Peptidemass mapping using a MALDI-TOF Mass spectrometer. The proteinconcentration was determined with Bradford Protein Assay.

Random Oligonucleotide Selection

According to the SELEX protocol described in Bouvet (2001) Methods MolBiol. 148:603-10, a 62 bp oligonucleotide having sequences as followswas synthesized: 5′-TGGGCACTATTTATATCAAC-N25-AATGTCGTTGGTGGCCC-3′ (SEQID NO: 244) where N is any nucleotide, and primers complementary to eachend. Primer P is: 5′-ACCGCAAGCTTGGGCACTATTTATATCAAC-3′ (SEQ ID NO: 245),and primer R is 5′-GGTCTAGAGGGCCACCAACGCATT-3′ (SEQ ID NO: 246). The62-mer oligonucleotide is made double stranded by PCR using the P and Rprimers generating a 80 bp random pool.

About 250 ng of THAP-His6 was incubated with Ni-NTA magnetic beads inNT2 buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% NP-40) for 30 minat 4° C. on a roller. The beads were washed 2 times with 500 μl of NT2buffer to remove unbound protein. The immobilized THAP-His6 wasincubated with the random pool of double stranded 80 bp DNA (2 to 5 μg)in 100 μl of Binding buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05%NP-40, 0.5 mM EDTA, 100 μg/ml BSA, and 20 to 50 μg of poly(dI-dC)) for10 minutes at room temperature. The beads were then washed 6 times with500 μl of NT2 buffer. The protein/DNA complex were then subjected toextraction with phenol/chloroform and precipitation with ethanol using10 μg of glycogen as a carrier. About one fifth of the recovered DNA wasthen amplified by 15 to 20 cycles of PCR and used for the next round ofselection. After 8 rounds of selection, the NaCl concentration wasprogressively increased to 150 mM.

After 12 rounds of selection by THAP-His6, pools of amplifiedoligonucleotides were digested with Xba I and Hind III and cloned intopBluescript II KS—(Stratagene) and individual clones were sequencedusing Big Dye terminator Kit (Applied Biosystem).

The results of the sequence analysis show that the THAP domain of humanTHAP1 is a site-specific DNA binding domain. Two consensus sequenceswere deduced from the alignment of two sets of nucleotide sequencesobtained from the above SELEX procedure (each set containing 9 nucleicacid sequences). In particular, it was found that the THAP domainrecognizes GGGCAA or TGGCAA DNA target sequences preferentiallyorganized as direct repeats with 5 nucleotide spacing (DR-5 motifs). Theconsensus sequence being GGGCAAnnnnnTGGCAA (SEQ ID NO: 149).Additionally, THAP recognizes everted repeats with 11 nucleotide spacing(ER-11 motifs) having a consensus sequence of TTGCCAnnnnnnnnnnnGGGCAA(SEQ ID NO: 159). Although GGGCAA and TGGCAA sequences constitute thepreferential THAP domain DNA binding sites, GGGCAT, GGGCAG and TGGCAGsequences are also DNA target sequences recognized by the THAP domain.

Example 29 High Throughput in vitro Assay to Identify Inhibitors ofTHAP-Family Polypeptide or THAP-Family Interactions with Nonspecific DNATargets

High throughput assays for the detection and quantification ofTHAP1-nonspecific DNA binding is carried out using a scintillationproximity assay. Materials are available from Amersham (Piscataway,N.J.) and assays can be carried out according to Gal S. et al, 6^(th)Ann. Conf. Soc. Biomol. Screening, 6-9 Sep. 2000, Vancouver, B.C.), thedisclosure of which is incorporated herein by reference in its entirety.

Random double stranded DNA probes are prepared and labelled using[³H]TTP and terminal transferase to a suitable specific activity (e.g.approx. 420 i/mmol). THAP1 protein or a portion thereof is prepared andthe quantity of THAP1 protein or a portion thereof is determined viaELISA. For assay development purposes, electrophoretic mobility shiftassays (EMSA) can be carried out to select suitable assay parameters.For the high throughput assay, ³H labelled DNA, anti-THAP1 monoclonalantibody and THAP1 in binding buffer (Hepes, pH 7.5; EDTA; DTT; 10 mMammonium sulfate; KCl and Tween-20) are combined. The assay isconfigured in a standard 96-well plate and incubated at room temperaturefor 5 to 30 minutes, followed by the addition of 0.5 to 2 mg of PVTprotein A SPA beads in 50-100 μl binding buffer. The radioactivity boundto the SPA beads is measured using a TopCount™ Microplate Counter(Packard Biosciences, Meriden, Conn.).

Example 30 High Throughput in vitro Assay to Identify Inhibitors ofTHAP-Family Polypeptide or THAP-Family Interactions with Specific DNATargets

High throughput assays for the detection and quantification of THAP1specific DNA binding is carried out using a scintillation proximityassay. Materials are available from Amersham (Piscataway, N.J.) andassays can be carried out according to Gal S. et al, 6^(th) Ann. Conf.Soc. Biomol. Screening, 6-9 Sep. 2000, Vancouver, B.C.).

THAP1-specific double stranded DNA probes corresponding to THAP1 DNAbinding sequences obtained according to Example 20 are prepared. Theprobes are labelled using [³H]TTP and terminal transferase to a suitablespecific activity (e.g. approx. 420i/mmol). THAP1 protein or a portionthereof is prepared and the quantity of THAP1 protein or a portionthereof is determined via ELISA. For assay development purposes,electrophoretic mobility shift assays (EMSA) can be carried out toselect suitable assay parameters. For the high throughput assay, 3Hlabelled DNA, anti-THAP1 monoclonal antibody, 1 μg non-specific DNA(double or single stranded poly-dAdT) and THAP1 protein or a portionthereof in binding buffer (Hepes, pH7.5; EDTA; DTT; 10 mM ammoniumsulfate; KCl and Tween-20) are combined. The assay is configured in astandard 96-well plate and incubated at room temperature for 5 to 30minutes, followed by the addition of 0.5 to 2 mg of PVT protein A SPAbeads in 50-100 μl binding buffer. The radioactivity bound to the SPAbeads is measured using a TopCount(m Microplate Counter (PackardBiosciences, Meriden, Conn.).

Example 31 Preparation of Antibody Compositions

Substantially pure THAP1 protein or a portion thereof is obtained. Theconcentration of protein in the final preparation is adjusted, forexample, by concentration on an Amicon filter device, to the level of afew micrograms per ml. Monoclonal or polyclonal antibodies to theprotein can then be prepared as follows: Monoclonal Antibody Productionby Hybridoma Fusion Monoclonal antibody to epitopes in the THAP1 proteinor a portion thereof can be prepared from murine hybridomas according tothe classical method of Kohler and Milstein (Nature, 256: 495, 1975) orderivative methods thereof (see Harlow and Lane, Antibodies A LaboratoryManual, Cold Spring Harbor Laboratory, pp. 53-242, 1988), the disclosureof which is incorporated herein by reference in its entirety.

Briefly, a mouse is repetitively inoculated with a few micrograms of theTHAP1 protein or a portion thereof over a period of a few weeks. Themouse is then sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and the excess unfused cells destroyed bygrowth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, E., Meth. Enzymol. 70: 419 (1980), the disclosure of which isincorporated herein by reference in its entirety. Selected positiveclones can be expanded and their monoclonal antibody product harvestedfor use. Detailed procedures for monoclonal antibody production aredescribed in Davis, L. et al. Basic Methods in Molecular Biology,Elsevier, New York., Section 21-2, the disclosure of which isincorporated herein by reference in its entirety.

Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes inthe THAP1 protein or a portion thereof can be prepared by immunizingsuitable non-human animal with the THAP1 protein or a portion thereof,which can be unmodified or modified to enhance immunogenicity. Asuitable nonhuman animal, preferably a non-human mammal, is selected.For example, the animal may be a mouse, rat, rabbit, goat, or horse.Alternatively, a crude protein preparation which, has been enriched forTHAP1 or a portion thereof can be used to generate antibodies. Suchproteins, fragments or preparations are introduced into the non-humanmammal in the presence of an appropriate adjuvant (e. g. aluminumhydroxide, RIBI, etc.) which is known in the art. In addition theprotein, fragment or preparation can be pretreated with an agent whichwill increase antigenicity, such agents are known in the art andinclude, for example, methylated bovine serum albumin (mBSA), bovineserum albumin (BSA), Hepatitis B surface antigen, and keyhole limpethemocyanin (KLH). Serum from the immunized animal is collected, treatedand tested according to known procedures. If the serum containspolyclonal antibodies to undesired epitopes, the polyclonal antibodiescan be purified by immunoaffinity chromatography.

Effective polyclonal antibody production is affected by many factorsrelated both to the antigen and the host species. Also, host animalsvary in response to site of inoculations and dose, with both inadequateor excessive doses of antigen resulting in low titer antisera. Smalldoses (ng level) of antigen administered at multiple intradermal sitesappears to be most reliable. Techniques for producing and processingpolyclonal antisera are known in the art, see for example, Mayer andWalker (1987), the disclosure of which is incorporated herein byreference in its entirety. An effective immunization protocol forrabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol.Metab. 33: 988-991 (1971), the disclosure of which is incorporatedherein by reference in its entirety. Booster injections can be given atregular intervals, and antiserum harvested when antibody titer thereof,as determined semi-quantitatively, for example, by doubleimmunodiffusion in agar against known concentrations of the antigen,begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in:Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973).Plateau concentration of antibody is usually in the range of 0.1 to 0.2mg/ml of serum (about 12: M). Affinity of the antisera for the antigenis determined by preparing competitive binding curves, as described, forexample, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2dEd. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).

Antibody preparations prepared according to either the monoclonal or thepolyclonal protocol are useful in quantitative immunoassays whichdetermine concentrations of antigen-bearing substances in biologicalsamples; or they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample. The antibodiesmay also be used in therapeutic compositions for killing cellsexpressing the protein or reducing the levels of the protein in thebody.

Example 32 Two Hybrid THAP1/Chemokine Interaction Assay

Two-hybrid interaction between THAP1 and chemokines CCL21, CCL19, CXCL9and CXCL10 or cytokine IFNγ was tested by cotransformation of AH109 withpGADT7-THAP1 and pGBKT7-CCL21, -CCL19, -CXCL9, -CXCL10 and -IFNγplasmids and selection of transformants by His and Ade double auxotrophyaccording to manufacturer's instructions (MATCHMAKER two-hybrid system3, Clontech). pGBKT7-chemokine vectors were generated using cDNAsencoding the mature forms of human chemokines CCL21 (see example 15)(SLC polypeptide SEQ ID NO: 271, SLC cDNA SEQ ID NO: 272); CCL19 (aminoacids 22-98 of GenBank Accession No. NM_(—)006274) (CCL19 polypeptideSEQ ID NO: 273, CCL19 cDNA SEQ ID NO: 274); CXCL9 (amino acids 23-125 ofGenBank Accession No. NM₁₃ 002416) (CXCL9 polypeptide SEQ ID NO: 275,CXCL9 cDNA SEQ ID NO: 276) CXCL10 (amino acids 22-98 of GenBankAccession No. NM_(—)001565) (CXCL10 polypeptide SEQ ID NO: 277, CXCL10cDNA SEQ ID NO: 278) or cytokine IFNγ (amino acids 21-166 of GenBankAccession No. NM_(—)000619) (IFNγ polypeptide SEQ ID NO: 279, IFNγ cDNASEQ ID NO: 280), amplified by PCR, respectively, from Image clones No.1707527 (hCCL19) with primers CCL19-1(5′-GCGGAATCATGGGCACCAATGATGCTGAAGACTG-3′) (SEQ ID NO: 281) and CCL19-2(5′-GCGGGATCCTTAACTGCTGCGGCGCTTCATCTTG-3′) (SEQ ID NO: 282), No. 5228247(hCXCL9) with primers CXCL9-1(5′-GCCGAATTCACCCCAGTAGTGAGAAAGGGTCGCTG-3′) (SEQ ID NO: 283) and CXCL9-2(5′-CGCGGATCCTTATGTAGTCTTCTTTTGACGAGAACGTTG-3′) (SEQ ID NO: 284), No.4274617 (hCXCL10) with primers CXCL10-1(5′-GCCGAATTCGTACCTCTCTCTAGAACCGTACGCTGT-3′) (SEQ ID NO. 285) andCXCL10-2 (5′-GCGGGATCCTTAAGGAGATCTTTTAGACATTTCCTTGCTA-3′) (SEQ ID NO.286), No. 2403734 (hIFNγ) with primers IFN-1(5′-GCGGAATCATGTGTTACTGCCAGGACCCATATG-3′) (SEQ ID NO: 287) and IFN-2(5′-GCGGGATCCTTACTGGGATGCTCTTCGACCTTG-3′) (SEQ ID NO: 288). The PCRproducts were digested with EcoRI and BamHI, and cloned between EcoRIand BamHI cloning sites of vector pGBKT7 (Clontech). Positive two-hybridinteraction of THAP1 was observed with chemokines CCL21, CCL19, andCXCL9 while chemokine CXCL10 gave an intermediate result (±) in thistwo-hybrid assay (see FIG. 19). The negative cytokine control, IFNγ, didnot have a positive interaction.

It will be appreciated that the above-described methods can be used todetermine whether any particular chemokine binds to any THAP-familypolypeptide. For example, cDNAs encoding THAP-family members THAP1 toTHAP11 as well as THAP0 from humans and other species can be cloned intoa first component vector of a two hybrid system. cDNAs encodingchemokines can be cloned into a second component vector of a two hybridsystem. The two vectors can be transformed into an appropriate yeaststrain, wherein the polypeptides are expressed and tested forinteraction. For example, chemokine CCL5 (polypeptide SEQ ID NO: 289,cDNA SEQ ID NO: 290) can be tested for interaction with full-lengthTHAP-1 or particular portions of THAP1, such as a nested deletionseries. Chemokines which can be tested for interaction with THAP-familyproteins include, but are not limited to, XCL1, XCL2, CCL1, CCL2, CCL3,CCL3L1, SCYA3L2, CCL4, CCL4L, CCL5, CCL6, CCL7, CCL8, SCYA9, SCYA10,CCL11, SCYA12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20,CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, clone 391, CARPCC-1, CCL1, CK-1, regakine-1, K203, CXCL1, CXCL1P, CXCL2, CXCL3, PF4,PF4V1, CXCL5, CXCL6, PPBP, SPBPBP, IL8, CXCL9, CXCL10, CXCL11, CXCL12,CXCL14, CXCL15, CXCL16, NAP-4, LFCA-1, Scyba, JSC, VHSV-induced protein,CX3CL1 and fCL1.

Example 33 In vitro THAP1/Chemokine Interaction Assay

To confirm the interaction observed in yeast two-hybrid system, weperformed in vitro GST pull down assays. THAP1, expressed as aGST-tagged fusion protein and immobilized on glutathione sepharose, wasincubated with radiolabeled chemokines that were translated in vitro.

To generate the GST-THAP1 expression vector, the full-length codingregion of THAP1 (a nucleic acid encoding amino acids 1-213 of THAP1) wasamplified by PCR from HEVEC cDNA with primers 2HMR8(5′-CGCGGATCCGTGCAGTCCTGCTCCGCCTACGGC-3′) (SEQ ID NO: 291 and 2HMR11(5′-CCGAATTCTTATGCTGGTACTTCAACTATTTCAAAGTAG-3′) (SEQ ID NO: 292),digested with BamHI and EcoRI, and cloned in frame downstream of theGlutathione S-Transferase ORF, between the BamHI and EcoRI sites of thepGEX-2T prokaryotic expression vector (Amersham Pharmacia Biotech,Saclay, France). The GST-THAP1 fusion protein encoded by plasmidpGEX-2T-THAP1 and the control GST protein encoded by plasmid pGEX-2T,were then expressed in E. Coli DH5α and purified by affinitychromatography with glutathione sepharose according to supplier'sinstructions (Amersham Pharmacia Biotech). The yield of proteins used inGST pull-down assays was determined by SDS-Polyacrylamide GelElectrophoresis (PAGE) and Coomassie blue staining analysis.

In vitro-translated chemokines were generated with the TNT-coupledreticulocyte lysate system (Promega, Madison, Wis., USA) using astemplates pGBKT7-CCL21, —CCL19, —CXCL9 and —CXCL10 chemokine vectors(see Example 32) or pCMV-SPORT6-CCL5 plasmid (Image clone No. 4185200).In vitro-translated IFNγ cytokine was generated similarly using astemplate plasmid pGBKT7-IFNγ. A 25 μl volume of ³⁵S-labelled chemokinewas incubated with immobilized GST-THAP1 or GST proteins overnight at 4°C., in the following binding buffer: 10 mM NaPO4 pH 8.0, 140 mM NaCl, 3mM MgCl2, 1 mM dithiothreitol (DTT), 0.05% NP40, and 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM Na vanadate, 50 mM β-glycerophosphate,25 μg/ml chymotrypsine, 5 μg/ml aprotinin, and 10 μg/ml leupeptin. Beadswere then washed 5 times in 1 ml binding buffer. Bound proteins wereeluted with 2× Laemmli SDS-PAGE sample buffer, fractionated by 10%SDS-PAGE and visualized by fluorography using Amplify (AmershamPharmacia Biotech). GST/THAP1 specifically bound to chemokines CCL21,CCL19, CCL5, CXCL9 and CXCL10 but not cytokine IFNγ (FIGS. 19 and 20).FIG. 19 shows that CCL21, CCL19, CCL5 and CXCL9 all strongly bound toimmobilized GST-THAP1 (indicated by +++ in the column labelled “In vitrobinding to GST-THAP1”). CXCL10 also bound to immobilized GST-THAP1(indicated by ++ in the column labelled “In vitro binding toGST-THAP1”). The cytokine IFNγ did not bind to immobilized GST-THAP1(indicated by—in the column labelled “In vitro binding to GST-THAP1”).Chemokines CCL21, CCL19, CCL5, CXCL9 and CXCL10 failed to interact withGST beads (negative control). FIG. 20 a shows that equivalent amounts ofchemokine or cytokine were tested in the in vitro GST-THAP1 bindingassays. FIG. 20 b shows that neither the cytokine, IFNγ, nor any of thechemokines bound to immobilized GST alone. FIG. 20 c shows thatchemokines, CXCL10, CXCL9 and CCL19, but not the cytokine IFNγ, bound toimmobilized GST-THAP1 fusions.

It will be appreciated that the above-described methods can be used todetermine whether any particular chemokine binds to any THAP-familypolypeptide. For example, cDNAs encoding THAP-family members THAP1 toTHAP11 as well as THAP0 from humans and other species can be cloned andexpressed as a GST fusion protein and immobilized to a solid support.cDNAs encoding chemokines can be translated in vitro and the resultingproteins incubated with the immobilized GST-THAP family fusions.Furthermore, a nested deletion series and/or point mutants of theTHAP-family polypeptides can also be generated as GST-fusions and testedto determine the exact location of the chemokine binding domain for anyTHAP-family polypeptide with respect to any chemokine. Chemokines whichcan be tested for binding to THAP-family proteins include, but are notlimited to, XCL1, XCL2, CCL1, CCL2, CCL3, CCL3L1, SCYA3L2, CCL4, CCL4L,CCL5, CCL6, CCL7, CCL8, SCYA9, SCYA10, CCL11, SCYA12, CCL13, CCL14,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCL26, CCL27, CCL28, clone 391, CARP CC-1, CCL1, CK-1,regakine-1, K203, CXCL1, CXCL1P, CXCL2, CXCL3, PF4, PF4V1, CXCL5, CXCL6,PPBP, SPBPBP, IL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14, CXCL15,CXCL16, NAP-4, LFCA-1, Scyba, JSC, VHSV-induced protein, CX3CL1 andfCL1.

Example 34 Chemotaxis Bioassay: Inhibition of CCL21/CCL19-InducedChemotaxis by THAP1 Oligomeric Forms

To demonstrate inhibition of CCL21/CCL19-induced chemotaxis by THAP1oligomers, fresh lymphocytes and a human cell line, each of whichexpresses the CCL21/CCL19 receptor CCR7, are assayed for a chemotacticresponse to chemokines in the presence or absence of oligomeric THAP1.Lymphocytes are purified from fresh heparinized human blood or mouselymph nodes and grown in RPMI 1640 glutamax I (Invitrogen GIBCO). HuT78cells are obtained from American Tissue Type Culture Collection(Accession Number TIB-161) and grown in Iscove's modified Dulbecco'smedium with 4 mM L-Glutamine adjusted to contain 1.5 g/l sodiumbicarbonate (Invitrogen GIBCO). Recombinant CCL21 and CCL19 humanchemokines are obtained from commercial suppliers (for example, R&D orChemicon).

Chemokine CCL21 or CCL19 is diluted in the appropriate culture mediumwithout serum at 20 ng/ml and 75 μl of this solution is transferred inappropriated wells of a 96-well microplate. Recombinant human THAP1oligomers (GST-THAP1 or Fc-THAP1 chimera) are serially diluted startingat 500 nM and 25 μl of the different dilutions are transferred inappropriate wells. Transwells are set carefully on each well and 100 μlof a cell suspension at 8.10⁶ cell/ml is added in the upper chamber.Following a 4-hour incubation at 37° C. and 5% CO₂, the cells which havemigrated to the lower chamber are quantified using the Celltiter Glosystem (Promega). A staining of the filter is also performed to verifythat no cells adhered to the lower side of the filter after themigration. The degree of CCL21/CCL19 induced chemotaxis inhibition byTHAP1 oligomeric forms is calculated by comparing the number of cellswhich have migrated in the presence or absence of the THAP1 oligomericforms.

Example 35 Inhibition of CCL21/CCL19-Induced Lymphocyte Adhesion toEndothelial Cells in vivo by THAP1 Oligomeric Forms

The capacity of THAP1 oligomeric forms to block the activity ofCCL21/CCL19 in vivo, including CCL21/CCL19-induced lymphocyte adhesionto endothelial cells, is assessed by measuring the ‘rolling/stickingphenotype’ of lymphocytes in mouse lymph nodes HEVs (High endothelialvenules) using intravital microscopy (microscopy on live animals) asdescribed in von Andrian (1996) Microcirculation (3):287-300; and vonAndrian UH, M'Rini C. (1998) Cell Adhes Commun. 6(2-3):85-96), thedisclosures of which are incorporated herein by reference in theirentireties. The rolling/sticking assay is performed as follows. Inbrief, the different steps of lymphocyte migration through HEVs(tethering, rolling, sticking, transendothelial migration) are analyzedby intravital microscopy in mice treated with recombinant human THAP1oligomers (GST-THAP1 or Fc-THAP1 chimera). For observation of lymphnodes, HEVs vessels (and adhesion processes occurring in these vessels)by intravital microscopy, a microsurgical exposition of the subiliac(superficial inguinal) lymph node is made on an anaesthetized mouse.Briefly, BALB/c mice (Charles River, St Germain sur l'Arbresle, France)are anesthetized by intraperitoneal injection of 5 mg/mL ketamine and 1mg/mL xylasine (10 mL/kg) and surgically prepared under astereomicroscope (Leica Microsystems SA, Rueil-Malmaison, France) toallow exposure of the node vessels. A catheter is inserted in thecontralateral femoral artery to permit subsequent retrograde injectionsof fluorescent cell suspensions or recombinant THAP1 oligomeric forms(QST-THAP1 or Fc-THAP1, 10-100 μg in 250 μl saline injected and allowedto bind for 5-30 min before injection of fluorescent cell suspensions).The mouse is then transferred to an intravital microscope (INM 100;Leica Microsystems SA). Body temperature is maintained at 37° C. using apadding heater. Lymph node vessels and fluorescent cells are observedthrough 10× or 20× water immersion objective (Leica Microsystems SA) bytransillumination or epifluorescence illumination. Transilluminated andfluorescent events are visualized using a silicon-intensified targetcamera (Hamamatsu Photonics, Massy, France) and recorded for lateroff-line analysis (DSR-11 Sony, IEC-ASV, Toulouse). Lymphocyte behaviorin lymph node vessels is analyzed off-line as previously described (vonAndrian (1996) Microcirculation (3):287-300; and von Andrian UH, M'RiniC. (1998) Cell Adhes Commun. 6(2-3):85-96). Briefly, the rollingfraction is determined in every visible lymph node HEV as the percentageof lymphocytes interacting with the endothelial lining over the totalcell number entering the venule during an observation period. Rollingcells that become subsequently adherent are included in the rollingfraction. The sticking fraction is determined as percentage of rollersthat becomes firmly adherent in HEVs for more than 20 seconds. Onlyvessels with more than 10 rolling cells are included. The degree ofinhibition of CCL21/CCL19-induced lymphocyte adhesion by THAP1 oligomersin vivo is calculated by comparing the number of lymphocytes sticking toendothelial cells (sticking fractions) in the presence or absence of theTHAP1 oligomeric forms.

Example 36 Use of THAP1 Oligomeric Forms to Antagonize Chemokines in aMouse Model of Rheumatoid Arthritis

This experiment is designed to test effect of antagonizing chemokineswith THAP1 oligomeric forms in a mouse model of rheumatoid arthritis,the well-known collagen-induced arthritis model. In each experiment,male DBA/1 mice are immunized with collagen on day 21 and are boosted onday 0. Mice are treated daily from days 0-14 with IP injections of THAP1oligomeric forms (GST-THAP1 or THAP1-Fc chimera) at 150, 50, 15, and 5μg/day, and compared to mice treated with control proteins (GST or humanIgG1), at 150 μg/day (n=15/group in each experiment). The incidence andseverity of arthritis is monitored in a blind study. Each paw isassigned a score from 0 to 4 as follows: 0=normal; 1=swelling in 1 to 3digits; 2=mild swelling in ankles, forepaws, or more than 3 digits;3=moderate swelling in multiple joints; 4=severe swelling with loss offunction. Each paw is totaled for a cumulative score/mouse. Thecumulative scores are then totaled for mice in each group for a meanclinical score. Groups of 15 mice are treated with the indicated dosesof THAP1-Fc or with 150 μg/day of human IgG1. The capacity of THAP1oligomeric forms (GST-THAP1 or THAP1-Fc chimera) to reduce the diseaseincidence and severity of arthritis is determined by comparison with thecontrol group.

Example 37 Use of THAP1 Olipomeric Forms to Antagonize Chemokines in aMouse of Inflammatory Bowel Disease

The effect of blocking chemokines with THAP1-Fc chimera is analyzed inan experimentally induced model of Inflammatory Bowel Disease (IBD). Oneof the most widely used models of IBD is the DSS model (dextran sulphatesalt). In this model, dextran sulphate salt (M.W. typically about 40,000but molecular weights from 40,000 to 500,000 can be used) is given tomice (or other small mammals) in their drinking water at 2% to 8%. Insome studies, C57BL/6 mice are given 2% DSS from day 0 to day 7 (D0-D7),wherein the number of mice per group equals four (n=4). The study groupsare divided as follows: No DSS+human IgG1 (250 μg/day/mouse D0-D7); 2%DSS+THAP1-Fc (100 μg/day/mouse D0-D7); 2% DSS+THAP1-Fc (250 μg/day/mouseD0-D7); 2% DSS+THAP1-Fc (500 μg/day/mouse D0-D7); 2% DSS+human IgG1 (250μg/day/mouse D0-D7). Mice are weighed daily. Weight loss is a clinicalsign of the disease. Tissues are harvested at day 8 (D8). Histopathologyis performed on the following tissues: small intestine, large intestineand mesenteric lymph nodes (MLN). The capacity of the THAP1-Fc chimera,to attenuate some of the weight loss associated with DSS-inducedcolitis, and to reduce inflammation in the large intestine is determinedby comparing mice treated with THAP1-Fc to mice treated with controlhuman IgG1.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

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1. A method of producing a nucleic acid that encodes an alteredchemokine binding domain of a THAP-family polypeptide, said methodcomprising synthesizing a nucleic acid which comprises a polynucleotidesequence having at least one nucleobase alteration as compared to apolynucleotide sequence encoding an unaltered chemokine binding domainof a THAP-family polypeptide, wherein said chemokine binding domainencoded by said altered nucleic acid retains the ability to bind atleast one chemokine.
 2. The method of claim 1, wherein said at least onenucleobase alteration comprises the addition of one or more nucleotidesas compared to the polynucleotide sequence encoding the unalteredchemokine binding domain of a THAP-family polypeptide.
 3. The method ofclaim 1, wherein said at least one nucleobase alteration comprises thedeletion of one or more nucleotides as compared to the polynucleotidesequence encoding the unaltered chemokine binding domain of aTHAP-family polypeptide.
 4. The method of claim 1, wherein said at leastone nucleobase alteration comprises one or more one nucleobasesubstitutions as compared to the polynucleotide sequence encoding theunaltered chemokine binding domain of a THAP-family polypeptide.
 5. Themethod of claim 1, wherein said at least one chemokine is a chemokinethat is bound by the unaltered chemokine binding domain of theTHAP-family polypeptide.
 6. The method of claim 1, wherein said at leastone chemokine is selected from the group consisting of XCL1, XCL2, CCL1,CCL2, CCL3, CCL3L1, SCYA3L2, CCL4, CCL4L, CCL5, CCL6, CCL7, CCL8, SCYA9,SCYA10, CCL11, SCYA12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, clone391, CARP CC-1, CCL1, CK-1, regakine-1, K203, CXCL1, CXCL1P, CXCL2,CXCL3, PF4, PF4V1, CXCL5, CXCL6, PPBP, SPBPBP, IL8, CXCL8, CXCL9,CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, NAP-4, LFCA-1,Scyba, JSC, VHSV-induced protein, CX3CL1 and fCL1.
 7. The method ofclaim 1, wherein said at least one chemokine is selected from the groupconsisting of SLC, CCL19, CCL5, CXCL9 and CXCL10.
 8. The method of claim1, wherein the chemokine specificity of the altered chemokine bindingdomain is unchanged as compared to the unaltered chemokine bindingdomain of the THAP-family polypeptide.
 9. The method of claim 1, whereinthe chemokine specificity of the altered chemokine binding domain ischanged as compared to the unaltered chemokine binding domain of theTHAP-family polypeptide.
 10. The method of claim 9, wherein the alteredchemokine binding domain binds at least one chemokine that does not bindto the unaltered chemokine binding domain of the THAP-familypolypeptide.
 11. The method of claim 9, wherein the altered chemokinebinding domain does not bind to at least one chemokine that binds to theunaltered chemokine binding domain of the THAP-family polypeptide. 12.The method of claim 1, wherein the affinity of chemokine binding to thealtered chemokine binding domain is unchanged as compared to theunaltered chemokine binding domain of the THAP-family polypeptide. 13.The method of claim 1, wherein the affinity of chemokine binding to thealtered chemokine binding domain is changed as compared to the unalteredchemokine binding domain of the THAP-family polypeptide.
 14. The methodof claim 13, wherein the altered chemokine binding domain binds at leastone chemokine that is bound by the unaltered chemokine binding domain ofthe THAP-family polypeptide more tightly than the unaltered chemokinebinding domain.
 15. The method of claim 13, wherein the alteredchemokine binding domain binds at least one chemokine that is bound bythe unaltered chemokine binding domain of the THAP-family polypeptideless tightly than the unaltered chemokine binding domain.
 16. The methodof claim 1, wherein the polynucleotide sequence having at least onenucleobase alteration as compared to a polynucleotide sequence encodingan unaltered chemokine binding domain of a THAP-family polypeptideremains at least 70% identical to the polynucleotide sequence encodingthe unaltered chemokine binding domain of the THAP-family polypeptide.17. The method of claim 1, wherein the polynucleotide sequence having atleast one nucleobase alteration as compared to a polynucleotide sequenceencoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 75% identical to the polynucleotidesequence encoding the unaltered chemokine binding domain of theTHAP-family polypeptide.
 18. The method of claim 1, wherein thepolynucleotide sequence having at least one nucleobase alteration ascompared to a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 80%identical to the polynucleotide sequence encoding the unalteredchemokine binding domain of the THAP-family polypeptide.
 19. The methodof claim 1, wherein the polynucleotide sequence having at least onenucleobase alteration as compared to a polynucleotide sequence encodingan unaltered chemokine binding domain of a THAP-family polypeptideremains at least 85% identical to the polynucleotide sequence encodingthe unaltered chemokine binding domain of the THAP-family polypeptide.20. The method of claim 1, wherein the polynucleotide sequence having atleast one nucleobase alteration as compared to a polynucleotide sequenceencoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 90% identical to the polynucleotidesequence encoding the unaltered chemokine binding domain of theTHAP-family polypeptide.
 21. The method of claim 1, wherein thepolynucleotide sequence having at least one nucleobase alteration ascompared to a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 95%identical to the polynucleotide sequence encoding the unalteredchemokine binding domain of the THAP-family polypeptide.
 22. The methodof claim 1, wherein the amino acid sequence encoded by thepolynucleotide—sequence having at least one nucleobase alteration ascompared a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 70%identical to the amino acid sequence of the unaltered chemokine bindingdomain of the THAP-family polypeptide.
 23. The method of claim 1,wherein the amino acid sequence encoded by the polynucleotide sequencehaving at least one nucleobase alteration as compared a polynucleotidesequence encoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 75% identical to the amino acid sequence ofthe unaltered chemokine binding domain of the THAP-family polypeptide.24. The method of claim 1, wherein the amino acid sequence encoded bythe polynucleotide sequence having at least one nucleobase alteration ascompared a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 80%identical to the amino acid sequence of the unaltered chemokine bindingdomain of the THAP-family polypeptide.
 25. The method of claim 1,wherein the amino acid sequence encoded by the polynucleotide sequencehaving at least one nucleobase alteration as compared a polynucleotidesequence encoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 85% identical to the amino acid sequence ofthe unaltered chemokine binding domain of the THAP-family polypeptide.26. The method of claim 1, wherein the amino acid sequence encoded bythe polynucleotide sequence having at least one nucleobase alteration ascompared a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 90%identical to the amino acid sequence of the unaltered chemokine bindingdomain of the THAP-family polypeptide.
 27. The method of claim 1,wherein the amino acid sequence encoded by the polynucleotide sequencehaving at least one nucleobase alteration as compared a polynucleotidesequence encoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 95% identical to the amino acid sequence ofthe unaltered chemokine binding domain of the THAP-family polypeptide.28. The method of claim 1, wherein the unaltered chemokine bindingdomain of the THAP-family polypeptide is a chemokine binding domain of aTHAP-family polypeptide, wherein the THAP-family polypeptide is selectedfrom the group consisting of THAP1 (SEQ ID NO: 3), THAP2 (SEQ ID NO: 4);THAP3 (SEQ ID NO: 5), THAP4 (SEQ ID NO: 6); THAP5 (SEQ ID NO: 7), THAP6(SEQ ID NO: 8); THAP7 (SEQ ID NO: 9), THAP8 (SEQ ID NO: 10); THAP9 (SEQID NO: 11), THAP10 (SEQ ID NO: 12); THAP11 (SEQ ID NO: 13) and THAP0(SEQ ID NO: 14).
 29. The method of claim 1, wherein the polynucleotidesequence encoding the unaltered chemokine binding domain of aTHAP-family polypeptide is a polynucleotide sequence which encodes thechemokine binding domain of a THAP-family polypeptide, wherein theTHAP-family polypeptide is encoded by a polynucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 160-171.
 30. The method ofclaim 1, further comprising translating the nucleic acid comprising apolynucleotide sequence having at least one nucleobase alteration ascompared to a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide, thereby producing apolypeptide comprising the altered chemokine binding domain.
 31. Themethod of claim 30, wherein the polypeptide comprising the alteredchemokine binding domain is a fusion of an IgFc region and the alteredchemokine binding domain.
 32. The method of claim 30, wherein thepolypeptide comprising the altered chemokine binding domain is amultimer of the altered chemokine binding domain.
 33. The method ofclaim 30, wherein the polypeptide comprising the altered chemokinebinding domain includes only the altered chemokine binding domain.
 34. Amethod of altering the nucleotide sequence of a nucleic acid thatencodes a chemokine binding domain of a THAP-family polypeptide, saidmethod comprising the steps of: (a) obtaining a nucleic acid comprisinga polynucleotide sequence that encodes a chemokine binding domain of aTHAP-family polypeptide; (b) generating from said nucleic acid, anucleic acid comprising a polynucleotide sequence having at least onenucleobase alteration as compared to a polynucleotide sequence encodingan unaltered chemokine binding domain of a THAP-family polypeptide,wherein said chemokine binding domain encoded by said altered nucleicacid retains the ability to bind at least one chemokine; and (c)optionally, repeating step (b) on the nucleic acid product of step (b).35. The method of claim 34 further comprising translating the nucleicacid comprising a polynucleotide sequence having at least one nucleobasealteration as compared to a polynucleotide sequence encoding anunaltered chemokine binding domain of a THAP-family polypeptide, therebyproducing a polypeptide comprising the altered chemokine binding domain.36. The method of claim 35 further comprising comparing the chemokinebinding activity of said polypeptide comprising the altered chemokinebinding domain to the chemokine binding activity of a polypeptidecomprising an unaltered chemokine binding domain or a polypeptidecomprising a chemokine binding domain having different alterations thansaid polypeptide comprising the altered chemokine binding domain. 37.The method of claim 34, wherein said at least one nucleobase alterationcomprises the addition of one or more nucleotides as compared to thepolynucleotide sequence encoding the unaltered chemokine binding domainof a THAP-family polypeptide.
 38. The method of claim 34, wherein saidat least one nucleobase alteration comprises the deletion of one or morenucleotides as compared to the polynucleotide sequence encoding theunaltered chemokine binding domain of a THAP-family polypeptide.
 39. Themethod of claim 34, wherein said at least one nucleobase alterationcomprises one or more one nucleobase substitutions as compared to thepolynucleotide sequence encoding the unaltered chemokine binding domainof a THAP-family polypeptide.
 40. The method of claim 34, wherein saidat least one chemokine is a chemokine that is bound by the unalteredchemokine binding domain of the THAP-family polypeptide.
 41. The methodof claim 34, wherein said at least one chemokine is selected from thegroup consisting of XCL1, XCL2, CCL1, CCL2, CCL3, CCL3L1, SCYA3L2, CCL4,CCL4L, CCL5, CCL6, CCL7, CCL8, SCYA9, SCYA10, CCL11, SCYA12, CCL13,CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCL26, CCL27, CCL28, clone 391, CARP CC-1, CCL1, CK-1,regakine-1, K203, CXCL1, CXCL1P, CXCL2, CXCL3, PF4, PF4V1, CXCL5, CXCL6,PPBP, SPBPBP, IL8, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, NAP-4, LFCA-1, Scyba, JSC, VHSV-induced protein, CX3CL1and fCL1.
 42. The method of claim 34, wherein said at least onechemokine is selected from the group consisting of SLC, CCL19, CCL5,CXCL9 and CXCL10.
 43. The method of claim 34, wherein the chemokinespecificity of the altered chemokine binding domain is unchanged ascompared to the unaltered chemokine binding domain of the THAP-familypolypeptide.
 44. The method of claim 34, wherein the chemokinespecificity of the altered chemokine binding domain is changed ascompared to the unaltered chemokine binding domain of the THAP-familypolypeptide.
 45. The method of claim 44, wherein the altered chemokinebinding domain binds at least one chemokine that does not bind to theunaltered chemokine binding domain of the THAP-family polypeptide. 46.The method of claim 44, wherein the altered chemokine binding domaindoes not bind to at least one chemokine that binds to the unalteredchemokine binding domain of the THAP-family polypeptide.
 47. The methodof claim 34, wherein the affinity of chemokine binding to the alteredchemokine binding domain is unchanged as compared to the unalteredchemokine binding domain of the THAP-family polypeptide.
 48. The methodof claim 34, wherein the affinity of chemokine binding to the alteredchemokine binding domain is changed as compared to the unalteredchemokine binding domain of the THAP-family polypeptide.
 49. The methodof claim 48, wherein the altered chemokine binding domain binds at leastone chemokine that is bound by the unaltered chemokine binding domain ofthe THAP-family polypeptide more tightly than the unaltered chemokinebinding domain.
 50. The method of claim 48, wherein the alteredchemokine binding domain binds at least one chemokine that is bound bythe unaltered chemokine binding domain of the THAP-family polypeptideless tightly than the unaltered chemokine binding domain.
 51. The methodof claim 34, wherein the polynucleotide sequence having at least onenucleobase alteration as compared to a polynucleotide sequence encodingan unaltered chemokine binding domain of a THAP-family polypeptideremains at least 70% identical to the polynucleotide sequence encodingthe unaltered chemokine binding domain of the THAP-family polypeptide.52. The method of claim 34, wherein the polynucleotide sequence havingat least one nucleobase alteration as compared to a polynucleotidesequence encoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 80% identical to the polynucleotidesequence encoding the unaltered chemokine binding domain of theTHAP-family polypeptide.
 53. The method of claim 34, wherein thepolynucleotide sequence having at least one nucleobase alteration ascompared to a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 90%identical to the polynucleotide sequence encoding the unalteredchemokine binding domain of the THAP-family polypeptide.
 54. The methodof claim 34, wherein the amino acid sequence encoded by thepolynucleotide sequence having at least one nucleobase alteration ascompared a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 70%identical to the amino acid sequence of the unaltered chemokine bindingdomain of the THAP-family polypeptide.
 55. The method of claim 34,wherein the amino acid sequence encoded by the polynucleotide sequencehaving at least one nucleobase alteration as compared a polynucleotidesequence encoding an unaltered chemokine binding domain of a THAP-familypolypeptide remains at least 80% identical to the amino acid sequence ofthe unaltered chemokine binding domain of the THAP-family polypeptide.56. The method of claim 34, wherein the amino acid sequence encoded bythe polynucleotide sequence having at least one nucleobase alteration ascompared a polynucleotide sequence encoding an unaltered chemokinebinding domain of a THAP-family polypeptide remains at least 90%identical to the amino acid sequence of the unaltered chemokine bindingdomain of the THAP-family polypeptide.
 57. The method of claim 34,wherein the unaltered chemokine binding domain of the THAP-familypolypeptide is a chemokine binding domain of a THAP-family polypeptide,wherein the THAP-family polypeptide is selected from the groupconsisting of THAP1 (SEQ ID NO: 3), THAP2 (SEQ ID NO: 4); THAP3 (SEQ IDNO: 5), THAP4 (SEQ ID NO: 6); THAP5 (SEQ ID NO: 7), THAP6 (SEQ ID NO:8); THAP7 (SEQ ID NO: 9), THAP8 (SEQ ID NO: 10); THAP9 (SEQ ID NO: 11),THAP10 (SEQ ID NO: 12); THAP11 (SEQ ID NO: 13) and THAP0 (SEQ ID NO:14).
 58. The method of claim 34, wherein the polynucleotide sequenceencoding the unaltered chemokine binding domain of a THAP-familypolypeptide is a polynucleotide sequence which encodes the chemokinebinding domain of a THAP-family polypeptide, wherein the THAP-familypolypeptide is encoded by a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs: 160-171.
 59. A method of producing aTHAP-family chemokine binding domain having an altered specificity oraffinity, said method comprising the steps of: (a) altering thenucleotide sequence of a nucleic acid comprising a polynucleotidesequence that encodes a chemokine binding domain of aTHAP-family-polypeptide, thereby generating an altered chemokine bindingdomain; (b) determining whether the specificity or chemokine bindingaffinity of said altered chemokine binding domain is changed as comparedto the chemokine binding domain prior to the alteration; (c) optionally,repeating steps (a) and (b); and (d) selecting the desired THAP-familychemokine binding domain having an altered specificity or affinity. 60.The method of claim 59, wherein said altered chemokine binding domaincomprises one or more additional amino acids as compared to thechemokine binding domain prior to the alteration.
 61. The method ofclaim 59, wherein said altered chemokine binding domain comprises atleast one amino acid deletion as compared to the chemokine bindingdomain prior to the alteration.
 62. The method of claim 59, wherein saidaltered chemokine binding domain comprises at least one amino acidsubstitution as compared to the chemokine binding domain prior to thealteration.
 63. The method of claim 59, wherein the altered chemokinebinding domain binds at least one chemokine that does not bind to thechemokine binding domain prior to the alteration.
 64. The method ofclaim 59, wherein the altered chemokine binding domain does not bind toat least one chemokine that binds to the chemokine binding domain priorto the alteration.
 65. The method of claim 59, wherein the alteredchemokine binding domain binds at least one chemokine that is bound bythe chemokine binding domain prior to the alteration more tightly thanthe unaltered chemokine binding domain.
 66. The method of claim 59,wherein the altered chemokine binding domain binds at least onechemokine that is bound by the chemokine binding domain prior to thealteration less tightly than the unaltered chemokine binding domain. 67.The method of claim 59, wherein the amino acid sequence of the alteredchemokine binding domain remains at least 70% identical to the aminoacid sequence of an unaltered chemokine binding domain of theTHAP-family polypeptide.
 68. The method of claim 59, wherein the aminoacid sequence of the altered chemokine binding domain remains at least80% identical to the amino acid sequence of an unaltered chemokinebinding domain of the THAP-family polypeptide.
 69. The method of claim59, wherein the amino acid sequence of the altered chemokine bindingdomain remains at least 90% identical to the amino acid sequence of anunaltered chemokine binding domain of the THAP-family polypeptide. 70.The method of claim 59, wherein the amino acid sequence of the alteredchemokine binding domain remains at least 95% identical to the aminoacid sequence of an unaltered chemokine binding domain of theTHAP-family polypeptide.
 71. The method of claim 59, the chemokinebinding domain prior to the alteration is a chemokine binding domain ofa THAP-family polypeptide, wherein the THAP-family polypeptide isselected from the group consisting of THAP 1 (SEQ ID NO: 3), THAP2 (SEQID NO: 4); THAP3 (SEQ ID NO: 5), THAP4 (SEQ ID NO: 6); THAP5 (SEQ ID NO:7), THAP6 (SEQ ID NO: 8); THAP7 (SEQ ID NO: 9), THAP8 (SEQ ID NO: 10);THAP9 (SEQ ID NO: 11), THAP10 (SEQ ID NO: 12); THAP11 (SEQ ID NO: 13)and THAP0 (SEQ ID NO: 14).
 72. A method of assessing the ability of apolypeptide to bind a chemokine, said method comprising: obtaining apolypeptide comprising an altered chemokine binding domain of aTHAP-family polypeptide, wherein said altered chemokine binding domaincomprises at least one amino acid alteration as compared to a chemokinebinding domain of a THAP-family polypeptide prior to alteration;contacting said altered chemokine binding domain with a chemokine; anddetermining whether said altered chemokine binding domain binds saidchemokine.
 73. The method of claim 72, wherein said chemokine bindingdomain of a THAP-family polypeptide prior to alteration is an unalteredchemokine binding domain of a THAP-family polypeptide.
 74. The method ofclaim 72, wherein said altered chemokine binding domain comprises one ormore additional amino acids as compared to the chemokine binding domainprior to the alteration.
 75. The method of claim 72, wherein saidaltered chemokine binding domain comprises at least one amino aciddeletion as compared to the chemokine binding domain prior to thealteration.
 76. The method of claim 72, wherein said altered chemokinebinding domain comprises at least one amino acid substitution ascompared to the chemokine binding domain prior to the alteration. 77.The method of claim 72, wherein the amino acid sequence of the alteredchemokine binding domain remains at least 70% identical to the aminoacid sequence of an unaltered chemokine binding domain of theTHAP-family polypeptide.
 78. The method of claim 72, wherein the aminoacid sequence of the altered chemokine binding domain remains at least80% identical to the amino acid sequence of an unaltered chemokinebinding domain of the THAP-family polypeptide.
 79. The method of claim72, wherein the amino acid sequence of the altered chemokine bindingdomain remains at least 90% identical to the amino acid sequence of anunaltered chemokine binding domain of the THAP-family polypeptide. 80.The method of claim 72, wherein the amino acid sequence of the alteredchemokine binding domain remains at least 95% identical to the aminoacid sequence of an unaltered chemokine binding domain of theTHAP-family polypeptide.
 81. The method of claim 72, the chemokinebinding domain prior to the alteration is a chemokine binding domain ofa THAP-family polypeptide, wherein the THAP-family polypeptide isselected from the group consisting of THAP1 (SEQ ID NO: 3), THAP2 (SEQID NO: 4); THAP3 (SEQ ID NO: 5), THAP4 (SEQ ID NO: 6); THAP5 (SEQ ID NO:7), THAP6 (SEQ ID NO: 8); THAP7 (SEQ ID NO: 9), THAP8 (SEQ ID NO: 10);THAP9 (SEQ ID NO: 11), THAP10 (SEQ ID NO: 12); THAP11 (SEQ ID NO: 13)and THAP0 (SEQ ID NO: 14).